Novel human leucine-rich repeat containing protein expressed predominately in small intestine, HLRRSI1

ABSTRACT

The present invention provides novel polynucleotides encoding HLRRSI1 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel HLRRSI1 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly gastrointestinal diseases and/or disorders. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

[0001] This application claims benefit to provisional application U.S.Ser. No. 60/257,774, filed Dec. 22, 2000.

FIELD OF THE INVENTION

[0002] The present invention provides novel polynucleotides encodingHLRRSI1 polypeptides, fragments and homologues thereof. Also providedare vectors, host cells, antibodies, and recombinant and syntheticmethods for producing said polypeptides. The invention further relatesto diagnostic and therapeutic methods for applying these novel HLRRSI1polypeptides to the diagnosis, treatment, and/or prevention of variousdiseases and/or disorders related to these polypeptides, particularlygastrointestinal diseases and/or disorders. The invention furtherrelates to screening methods for identifying agonists and antagonists ofthe polynucleotides and polypeptides of the present invention.

BACKGROUND OF THE INVENTION

[0003] Recently, a class of cell surface proteins have been described inboth plants and animals that are involved in pathogen perception, MHCclass II trans-activation, inflammation and the regulation of apoptosis(Inohara, N., Nunez, G, Cell, Death, Differ., 6(9):823-4, (1999);Inohara, N., Koseki, T., del, Peso, L., Hu, Y., Yee, C., Chen, S.,Carrio, R., Merino, J., Liu, D., Ni, J., Nunez, G, J. Biol, Chem. 21.,274(21):14560-7, (1999); Inohara, N., Nunez, G, Cell, Death, Differ.,7(5):509-10, (2000); Harton, J A., Ting, J P, Mol, Cell, Biol.,20(17):6185-94, (2000); Dixon, J., Brakebusch, C., Fassler, R., Dixon, MJ. Hum, Mol, Genet. 12., 9(10):1473-80, (2000)). All of these proteinsare modular in nature containing one or several domains that function incaspase recuritment (CARD), nucleotide binding and protein-proteininteractions. Proteins within this group have also been found to play arole in cell adhesion during various developmental processes.

[0004] A common theme in all of these proteins are the presence of aleucine-repeat repeat (LLR) in the carboxy terminus of the polypeptidechain. LLRs are short protein modules characterized by a periodicdistribution of hydrophobic amino acids, especially leucine residuesseparated by hydrophilic residues [Sean, 1996]. The basic structure ofthe repeat is as follows: X-L-X-X-L-X-L-X-X-N-X-a-X-X-X-a-X-X-L-X

[0005] where X is any amino acid, L is leucine, N is asparagine and “a”denotes an aliphatic residue. The asparagine at position 10 can bereplaced by cysteine, threonine or glutamine. The average repeat lengthis 24 amino acids but it can vary between 22 to 29 amino acids, thoughsome LRR motifs have been reported to be at short as 20 amino acids. Themotif often consists of leucine or other aliphatic residues at positions2, 5, 7, 12, 16, 21, and 24 and asparagine, cysteine or threonine atposition 10. X-ray structure determination of LRR motifs suggests thateach LRR is composed of a beta-sheet and an alpha-helix. The largestsubfamily of proteins that contain a leucine-rich domain areextracellular proteins having the following motif:LxxLxxLxLxxNxLxxLPxxOFxx, where “x” is any amino acid and “O” is anon-polar residue (Kajava, J. Mol. Biol. 277: 519 (1998)).

[0006] In transmembrane proteins, LLRs and their flanking sequencealways occur in the presumed extracellular portions. In these situationsthe LLRs are generally flanked on either side by cysteine-rich regions.In general, these cysteines are present in the oxidized disulphide linkform. An example of a transmembrane protein containing a LRR is Toll, aDrosophila gene the functions in establishment of dorsal-ventralpatterning. Dominant, ventralizing mutants have been described that mapto the cysteine-rich regions surrounding the LLR domain [Schneider,1991]. Thus, the cysteine regions associated with LLRs act to regulatereceptor activity. The LLRs themselves within the Toll protein have beenshown to function in heterotypic cell adhesion, a process required forproper motoneuron and muscle development [Halfon, 1995]

[0007] Another Drosophila LLR containing transmembrane protein, 18wheeler, which is regulated by homeotic genes also promotes heterophiliccell adhesion in cell migration events during development (Eldon, E.,Kooyer, S., D'Evelyn, D., Duman, M., Lawinger, P., Botas, J., Bellen, H,Development., 120(4):885-99, (1994)). Mammalian CD14, which bindslipopolysaccharide (LPS), and signals through NF-κB, is thought to haveanalogies to the Toll signal transduction pathway. CD14 also contains aregion of LLRs that have been shown in deletion mutants to beresponsible for LPS binding.

[0008] Slit is another LLR containing Drosophila secreted protein thatfunctions in the development of the midline glial cells and thecommissural axon tracts the cross the midline. This is presumablyaccomplished by cell adhesion events (Jacobs, J R, J. Neurobiol.,24(5):611-26, (1993)). Mammalian homologues of Drosophila slit have beenshown to bind the heparan sulfate proteoglycan, glypican-1 (Liang, Y.,Annan, R S., Carr, S A., Popp, S., Mevissen, M., olis, R K., olis, R U,J. Biol, Chem. 18., 274(25): 17885-92, (1999)). In general, heparansulfate proteoglycans have been shown to accummulate in Alzheimer'sdisease brains and specifically, glypican-1 is component of both senileplaques and neurofibrillary tangles (Verbeek, M M., Otte, Holler, I.,van, den, Born, J., van, den, Heuvel, L P., David, G., Wesseling, P.,de, Waal, R M, Am. J. Pathol., 155(6):2115-25, (1999)). Heparan sulfateproteoglycans are also implicated in the regulation of cytokinesignaling in B cells through the activation of CD40 (van, der, Voort,R., Taher, T E., Derksen, P W., Spaargaren, M., van, der, Neut, R.,Pals, S T, Adv, Cancer, Res., 79:39-90, (2000)).

[0009] p37NB is a 37 kDa LRR protein identified in human neuroblastomacells (Kim, D. et al. (1996) Biochim. Biophys. Acta 1309: 183-188).Northern blot hybridization and RT-PCR studies show that p37NB isdifferentially expressed in several neuroblastoma cell lines. A relatedLRR protein, PRELP, is characterized as a 42 kDa secreted protein(Bengtsson, E. et al. (1995) J. Biol. Chem. 270: 25639-25644). PRELPconsists of 10 LRR motifs ranging in length from 20 to 26 residues withasparigine at position 10. Northern analysis shows differentialexpression of PRELP in various tissues.

[0010] In addition, leucine-rich repeat containing proteins have alsobeen implicated in various aspects of protein-protein interaction, suchas cell-to-cell communication and signal transduction (for a review, seeKobe and Deisenhofer, TIBS 19: 415 (1994); Kobe and Deisenhofer, Curr.Opin. Struct. Biol. 5: 409 (1995); Kajava, J. Mol. Biol. 277: 519(1998)). Proteins that contain an LRR motif include hormone receptors,enzyme subunits, cell adhesion proteins, and ribosome-binding proteins.

[0011] A subfamily of the LRR superfamily, referred to as the SmallLeucine Rich Proteoglycan family, illustrates the critical functionsfulfilled by proteins containing an LRR motif. Members of this subfamilyare believed to play essential biological roles during inflammation andcancer invasion, a regulatory role in collagen fibril formation,suppression of the malignant phenotype of cancer cells, and aninhibition of the growth of certain normal cells (see, for example,Iozzo, Annu. Rev Biochem. 67: 609 (1998)).

[0012] Kajava, et al., J. Mol. Biol. 277: 519 (1998), divided the LRRsuperfamily into subfamilies characterized by different lengths andconsensus sequences of the leucine-rich repeats. Based upon thisstructural analysis, Kajava concluded that LRR proteins of differentsubfamilies probably emerged independently during evolution, indicatingthat proteins with the LRR motif provide a unique solution for a widerange of biological functions.

[0013] LLR containing proteins have been identified in prokaryotes,plants, yeast and mammals. Although such proteins were initially thoughtto be secreted proteins, it is now appreciated that they inhibit avariety of cellular locations and participate in a diverse set ofcritical functions in development and cellular homeostasis.

[0014] Such LRRs, being extracellular, are capable of directingprotein-protein interactions with other receptors involved in apoptosis,inflammation and immune responses. LLR containing proteins may also bindother extracellular ligands derived from infectious agents andparticipate in the triggering and or modulating immune responses,particularly apoptosis.

[0015] The mechanisms that mediate apoptosis have been intensivelystudied. These mechanisms involve the activation of endogenousproteases, loss of mitochondrial function, and structural changes suchas disruption of the cytoskeleton, cell shrinkage, membrane blebbing,and nuclear condensation due to degradation of DNA.

[0016] The various signals that trigger apoptosis are thought to bringabout these events by converging on a common cell death pathway, thecore components of which are highly conserved from worms, such as C.elegans, to humans. In fact, invertebrate model systems have beeninvaluable tools in identifying and characterizing the genes thatcontrol apoptosis. Despite this conservation of certain core components,apoptotic signaling in mammals is much more complex than ininvertebrates. For example, in mammals there are multiple homologues ofthe core components in the cell death signaling pathway.

[0017] Caspases, a class of proteins central to the apoptotic program,are responsible for the degradation of cellular proteins that leads tothe morphological changes seen in cells undergoing apoptosis. Caspases(cysteinyl aspartate-specific proteinases) are cysteine proteases havingspecificity for aspartate at the substrate cleavage site. Generally,caspases are classified as either initiator caspases or effectorcaspases, both of which are zymogens that are activated by proteolysisthat generates an active species. An effector caspase is activated by aninitiator caspase which cleaves the effector caspase.

[0018] Initiator caspases are activated by an autoproteolytic mechanismthat is often dependent upon oligomerization directed by association ofthe caspase with an adapter molecule.

[0019] Apoptotic signaling is dependent on protein-protein interactions.At least three different protein-protein interaction domains, the deathdomain, the death effector domain and the caspase recruitment domain(CARD), have been identified within proteins involved in apoptosis. Afourth protein-protein interaction domain, the death recruiting domain(DRD) was recently identified in murine FLASH (Imai et al. (1999) Nature398: 777-85).

[0020] Caspases comprise a multi-gene family having at least 12 distinctfamily members (Nicholson (1999) Cell Death and Differentiation 6:1028). A relatively small fraction of cellular polypeptides (less than200) are thought to serve as targets for cleavage by caspases. Becausemany of these caspase targets perform key cellular functions, theirproteolysis is thought to account for the cellular and morphologicalevents that occur during apoptosis. Members of the caspase gene familycan be divided by phylogenetic analysis into two major subfamilies,based upon their relatedness to ICE (interleukin-1p converting enzyme;caspase-1) and CED-3. Alternate groupings of caspases can be made basedupon their substrate specificities. Many caspases and proteins thatinteract with caspases possess a CARD domain.

[0021] The fate of a cell in multicellular organisms often requireschoosing between life and death. This process of cell suicide, known asprogrammed cell death or apoptosis, occurs during a number of events inan organisms life cycle, such as for example, in development of anembryo, during the course of an immunological response, or in the demiseof cancerous cells after drug treatment, among others. The final outcomeof cell survival versus apoptosis is dependent on the balance of twocounteracting events, the onset and speed of caspase cascade activation(essentially a protease chain reaction), and the delivery ofantiapoptotic factors which block the caspase activity (Aggarwal B. B.Biochem. Pharmacol. 60, 1033-1039, (2000); Thornberry, N. A. andLazebnik, Y. Science 281, 1312-1316, (1998)).

[0022] The production of antiapoptotic proteins is controlled by thetranscriptional factor complex NF-kB. For example, exposure of cells tothe protein tumor necrosis factor (TNF) can signal both cell death andsurvival, an event playing a major role in the regulation ofimmunological and inflammatory responses (Ghosh, S., May, M. J., Kopp,E. B. Annu. Rev. Immunol. 16, 225-260, (1998); Silverman, N. andManiatis, T., Genes & Dev. 15, 2321-2342, (2001); Baud, V. and Karin,M., Trends Cell Biol. 11, 372-377, (2001)). The anti-apoptotic activityof NF-kB is also crucial to oncogenesis and to chemo- andradio-resistance in cancer (Baldwin, A. S., J. Clin. Inves. 107,241-246, (2001)).

[0023] Nuclear Factor-kB (NF-kB), is composed of dimeric complexes ofp50 (NF-kB1) or p52 (NF-kB2) usually associated with members of the Relfamily (p65, c-Rel, Rel B) which have potent transactivation domains.Different combinations of NF-kB/Rel proteins bind distinct kB sites toregulate the transcription of different genes. Early work involvingNF-kB suggested its expression was limited to specific cell types,particularly in stimulating the transcription of genes encoding kappaimmunoglobulins in B lymphocytes. However, it has been discovered thatNF-kB is, in fact, present and inducible in many, if not all, cell typesand that it acts as an intracellular messenger capable of playing abroad role in gene regulation as a mediator of inducible signaltransduction. Specifically, it has been demonstrated that NF-kB plays acentral role in regulation of intercellular signals in many cell types.For example, NF-kB has been shown to positively regulate the humanbeta-interferon (beta-IFN) gene in many, if not all, cell types.Moreover, NF-kB has also been shown to serve the important function ofacting as an intracellular transducer of external influences.

[0024] The transcription factor NF-kB is sequestered in an inactive formin the cytoplasm as a complex with its inhibitor, IkB, the mostprominent member of this class being IkBa. A number of factors are knownto serve the role of stimulators of NF-kB activity, such as, forexample, TNF. After TNF exposure, the inhibitor is phosphorylated andproteolytically removed, releasing NF-kB into the nucleus and allowingits transcriptional activity. Numerous genes are upregulated by thistranscription factor, among them IkBa. The newly synthezised IkBaprotein inhibits NF-kB, effectively shutting down furthertranscriptional activation of its downstream effectors. However, asmentioned above, the IkBa protein may only inhibit NF-kB in the absenceof IkBa stimuli, such as TNF stimulation, for example. Other agents thatare known to stimulate NF-kB release, and thus NF-kB activity, arebacterial lipopolysaccharide, extracellular polypeptides, chemicalagents, such as phorbol esters, which stimulate intracellularphosphokinases, inflammatory cytokines, IL-1, oxidative and fluidmechanical stresses, and Ionizing Radiation (Basu, S., Rosenzweig, K,R., Youmell, M., Price, B, D, Biochem, Biophys, Res, Commun.,247(1):79-83, (1998)). Therefore, as a general rule, the stronger theinsulting stimulus, the stronger the resulting NF-kB activation, and thehigher the level of IkBa transcription. As a consequence, measuring thelevel of IkBa RNA can be used as a marker for antiapoptotic events, andindirectly, for the onset and strength of pro-apoptotic events.

[0025] Using the above examples, it is clear the availability of a novelcloned leucine-rich repeat containing protein provides an opportunityfor adjunct or replacement therapy, and are useful for theidentification of leucine-rich repeat containing protein agonists, orstimulators (which might stimulate and/or bias leucine-rich repeatcontaining protein action), as well as, in the identification ofleucine-rich repeat containing protein inhibitors. Hence it can bereasoned that agonists and antagonists for these LLR containing proteinswill be useful for therapeutic purposes

[0026] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells, in addition to their usein the production of HLRRSI1 polypeptides or peptides using recombinanttechniques. Synthetic methods for producing the polypeptides andpolynucleotides of the present invention are provided. Also provided arediagnostic methods for detecting diseases, disorders, and/or conditionsrelated to the HLRRSI1 polypeptides and polynucleotides, and therapeuticmethods for treating such diseases, disorders, and/or conditions. Theinvention further relates to screening methods for identifying bindingpartners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

[0027] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe HLRRSI1 protein having the amino acid sequence shown in FIGS. 1A-C(SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone,HLRRSI1 (also referred to as GPCR12#99, GPCR12#100, SILL1A and/orSILL1B), deposited as ATCC Deposit Number PTA-2679, and PTA-2674 on Nov.15, 2000.

[0028] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells, in addition to their usein the production of HLRRSI1 polypeptides or peptides using recombinanttechniques. Synthetic methods for producing the polypeptides andpolynucleotides of the present invention are provided. Also provided arediagnostic methods for detecting diseases, disorders, and/or conditionsrelated to the HLRRSI1 polypeptides and polynucleotides, and therapeuticmethods for treating such diseases, disorders, and/or conditions. Theinvention further relates to screening methods for identifying bindingpartners of the polypeptides.

[0029] The invention further provides an isolated HLRRSI1 polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0030] The invention further relates to a polynucleotide encoding apolypeptide fragment of SEQ ID NO:2, or a polypeptide fragment encodedby the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1.

[0031] The invention further relates to a polynucleotide encoding apolypeptide domain of SEQ ID NO:2 or a polypeptide domain encoded by thecDNA sequence included in the deposited clone, which is hybridizable toSEQ ID NO:1.

[0032] The invention further relates to a polynucleotide encoding apolypeptide epitope of SEQ ID NO:2 or a polypeptide epitope encoded bythe cDNA sequence included in the deposited clone, which is hybridizableto SEQ ID NO:1.

[0033] The invention further relates to a polynucleotide encoding apolypeptide of SEQ ID NO:2 or the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, having biologicalactivity.

[0034] The invention further relates to a polynucleotide which is avariant of SEQ ID NO:1.

[0035] The invention further relates to a polynucleotide which is anallelic variant of SEQ ID NO:1.

[0036] The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:2.

[0037] The invention further relates to a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:1.

[0038] The invention further relates to a polynucleotide capable ofhybridizing under stringent conditions to any one of the polynucleotidesspecified herein, wherein said polynucleotide does not hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence of only A residues or of only T residues.

[0039] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:2, wherein the polynucleotide fragment comprises anucleotide sequence encoding a leucine-rich repeat protein.

[0040] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:1, wherein the polynucleotide fragment comprises anucleotide sequence encoding the sequence identified as SEQ ID NO:2 orthe polypeptide encoded by the cDNA sequence included in the depositedclone, which is hybridizable to SEQ ID NO:1.

[0041] The invention further relates to an isolated nucleic acidmolecule of of SEQ ID NO:1, wherein the polynucleotide fragmentcomprises the entire nucleotide sequence of SEQ ID NO:1 or the cDNAsequence included in the deposited clone, which is hybridizable to SEQID NO:1.

[0042] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:1, wherein the nucleotide sequence comprisessequential nucleotide deletions from either the C-terminus or theN-terminus.

[0043] The invention further relates to an isolated polypeptidecomprising an amino acid sequence that comprises a polypeptide fragmentof SEQ ID NO:2 or the encoded sequence included in the deposited clone.

[0044] The invention further relates to a polypeptide fragment of SEQ IDNO:2 or the encoded sequence included in the deposited clone, havingbiological activity.

[0045] The invention further relates to a polypeptide domain of SEQ IDNO:2 or the encoded sequence included in the deposited clone.

[0046] The invention further relates to a polypeptide epitope of SEQ IDNO:2 or the encoded sequence included in the deposited clone.

[0047] The invention further relates to a full length protein of SEQ IDNO:2 or the encoded sequence included in the deposited clone.

[0048] The invention further relates to a variant of SEQ ID NO:2.

[0049] The invention further relates to an allelic variant of SEQ IDNO:2. The invention further relates to a species homologue of SEQ IDNO:2.

[0050] The invention further relates to the isolated polypeptide of ofSEQ ID NO:2, wherein the full length protein comprises sequential aminoacid deletions from either the C-terminus or the N-terminus.

[0051] The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:2.

[0052] The invention further relates to a method for preventing,treating, or ameliorating a medical condition, comprising administeringto a mammalian subject a therapeutically effective amount of thepolypeptide of SEQ ID NO:2 or the polynucleotide of SEQ ID NO:1.

[0053] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence orabsence of a mutation in the polynucleotide of SEQ ID NO:1; and (b)diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

[0054] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence oramount of expression of the polypeptide of of SEQ ID NO:2 in abiological sample; and diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.

[0055] The invention further relates to a method for identifying abinding partner to the polypeptide of SEQ ID NO:2 comprising the stepsof (a) contacting the polypeptide of SEQ ID NO:2 with a binding partner;and (b) determining whether the binding partner effects an activity ofthe polypeptide.

[0056] The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:1.

[0057] The invention further relates to a method of identifying anactivity in a biological assay, wherein the method comprises the stepsof expressing SEQ ID NO:1 in a cell, (b) isolating the supernatant; (c)detecting an activity in a biological assay; and (d) identifying theprotein in the supernatant having the activity.

[0058] The invention further relates to a process for makingpolynucleotide sequences encoding gene products having altered SEQ IDNO:2 activity comprising the steps of (a) shuffling a nucleotidesequence of SEQ ID NO:1, (b) expressing the resulting shufflednucleotide sequences and, (c) selecting for altered activity as comparedto the activity of the gene product of said unmodified nucleotidesequence.

[0059] The invention further relates to a shuffled polynucleotidesequence produced by a shuffling process, wherein said shuffled DNAmolecule encodes a gene product having enhanced tolerance to aninhibitor of SEQ ID NO:2 activity.

[0060] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, in addition to, its encoding nucleic acid,wherein the medical condition is a renal disorder

[0061] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, in addition to, its encoding nucleic acid,wherein the medical condition is a neural disorder.

[0062] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, in addition to, its encoding nucleic acid,wherein the medical condition is a disorder related to aberrant calciumregulation.

[0063] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, in addition to, its encoding nucleic acid,wherein the medical condition is a reproductive disorder.

[0064] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, in addition to, its encoding nucleic acid,wherein the medical condition is a neural disorder.

[0065] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, in addition to, its encoding nucleic acid,wherein the medical condition is a renal disorder.

[0066] The invention further relates to a method of identifying acompound that modulates the biological activity of HLRRSI1, comprisingthe steps of, (a) combining a candidate modulator compound with HLRRSI1having the sequence set forth in one or more of SEQ ID NO:2; andmeasuring an effect of the candidate modulator compound on the activityof HLRRSI1.

[0067] The invention further relates to a method of identifying acompound that modulates the biological activity of a leucine-rich repeatprotein, comprising the steps of, (a) combining a candidate modulatorcompound with a host cell expressing HLRRSI1 having the sequence as setforth in SEQ ID NO:2; and, (b) measuring an effect of the candidatemodulator compound on the activity of the expressed HLRRSI1.

[0068] The invention further relates to a method of identifying acompound that modulates the biological activity of HLRRSI1, comprisingthe steps of, (a) combining a candidate modulator compound with a hostcell containing a vector described herein, wherein HLRRSI1 is expressedby the cell; and, (b) measuring an effect of the candidate modulatorcompound on the activity of the expressed HLRRSI1.

[0069] The invention further relates to a method of screening for acompound that is capable of modulating the biological activity ofHLRRSI1, comprising the steps of: (a) providing a host cell describedherein; (b) determining the biological activity of HLRRSI1 in theabsence of a modulator compound; (c) contacting the cell with themodulator compound; and (d)determining the biological activity ofHLRRSI1 in the presence of the modulator compound; wherein a differencebetween the activity of HLRRSI1 in the presence of the modulatorcompound and in the absence of the modulator compound indicates amodulating effect of the compound.

[0070] The invention further relates to a compound that modulates thebiological activity of human HLRRSI1 as identified by the methodsdescribed herein.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

[0071] FIGS. 1A-C show the polynucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of the novel humanleucine-rich repeat containing protein, HLRRSI1, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 2689 nucleotides (SEQ ID NO:1), encodinga polypeptide of 625 amino acids (SEQ ID NO:2). An analysis of theHLRRSI1 polypeptide determined that it comprised the following features:three transmembrane domains located from about amino acid 38 to aboutamino acid 61, from about amino acid 115 to about amino acid 131, and/orfrom about amino acid 151 to about amino acid 167 of SEQ ID NO:2 (FIGS.1A-C) represented by underlining; conserved leucine residues located atamino acid 2, 9, 42, 45, 46, 51, 57, 68, 115, 118, 132, 154, 176, 179,182, 199, 247, 264, 284, 356, 359, 408, 424, 444, 463, 485, 494, 496,509, 537, 541, 547, 552, 557, 565, 569, 576, 579, 581, 586, 594, and 604of SEQ ID NO:2 represented by shading; differentially conserved leucineresidues located at amino acid 8, 12, 56, 161, 204, 219, 240, 243, 310,406, 428, 466, 491, and 550 of SEQ ID NO:2 represented in bold; andconserved cysteine residues located at amino acid 29, 129, 299, 358,396, 461, 518, and 574 of SEQ ID NO:2 represented by double underlining.The conserved leucine residues are characteristic of leucine-rich repeatproteins as described more particularly elsewhere herein. The conservedcysteine residues are diagnostic of conserved structural features of theprotein to leucine-rich repeat containing proteins (particularly thosereferenced herein), and may be indicative of conserved protein function.

[0072] FIGS. 2A-C shows the regions of identity between the encodedHLRRSI1 protein (SEQ ID NO:2) to other leucine-rich repeat proteins,specifically, the human caspase recruitment protein 7 protein(caspase_recruitment_protein; Genbank Accession No:gi|10198209; SEQ IDNO:3); the human nucleotide binding site protein protein(Nucleotide_Binding_site; Genbank Accession No:gi|10198207; SEQ IDNO:4); and the human cryopyrin protein (cryopyrin; Genbank AccessionNo:gi|17027237; SEQ ID NO:33). The alignment was performed using theCLUSTALW algorithm using default parameters as described herein (VectorNTI suite of programs). The darkly shaded amino acids represent regionsof matching identity. The lightly shaded amino acids represent regionsof matching similarity. Dots (“·”) between residues indicate gappedregions of non-identity for the aligned polypeptides. The conservedleucine residues between HLRRSI1 and the other leucine-rich repeatcontaining proteins are noted.

[0073]FIG. 3 shows a hydrophobicity plot of HLRRSI1 according to theBioPlot Hydrophobicity algorithm of Vector NTI (version 5.5). The threeputative transmembrane domains of the HLRRSI1 polypeptide are shown.

[0074]FIG. 4 shows an expression profile of the novel leucine-richrepeat containing protein, HLRRSI1. The figure illustrates the relativeexpression level of HLRRSI1 amongst various mRNA tissue sources. Asshown, transcripts corresponding to HLRRSI1 expressed predominately highin the small intestine, and to a lesser extent, in liver, lymph node,and spleen. Expression data was obtained by measuring the steady stateHLRRSI1 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:11 and 12 as described herein.

[0075]FIG. 5 shows a table illustrating the percent identity and percentsimilarity between the HLRRSI1 polypeptide of the present invention withother leucine-rich repeat containing proteins, specifically, the humancaspase recruitment protein 7 protein (caspase_recruitment_protein;Genbank Accession No:gi|10198209; SEQ ID NO:3); the human nucleotidebinding site protein protein (Nucleotide_Binding_site; Genbank AccessionNo:gi|10198207; SEQ ID NO:4); and the human cryopyrin protein(cryopyrin; Genbank Accession No:gi|17027237; SEQ ID NO:33). The percentidentity and percent similarity values were determined using the Gapalgorithm using default parameters (Genetics Computer Group suite ofprograms; Needleman and Wunsch. J. Mol. Biol. 48; 443-453, 1970)).

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein.

[0077] The invention provides a novel human sequence that encodes anovel leucine-rich repeat containing protein, HLRRSI1, with substantialhomology to the class of leucine-rich repeat containing proteins knownas caspase recruitment proteins. Members of this class of leucine-richrepeat proteins have been implicated in a number of diseases and/ordisorders, which include, but are not limited to, apoptosis andinflammatory disorders. Expression analysis indicates the HLRRSI1 hasstrong preferential expression in small intestine, and to a lesserextent, in liver, lymph node, and spleen. Based on this information, wehave provisionally named the gene and protein HLRRSI1 (HumanLeucine-Rich Repeat Small Intestine-1). The specificity by which theHLRRSI1 transcript is expressed that suggests its importance in variousbiological processes.

[0078] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide. The term “isolated” does not refer to genomic or cDNAlibraries, whole cell total or mRNA preparations, genomic DNApreparations (including those separated by electrophoresis andtransferred onto blots), sheared whole cell genomic DNA preparations orother compositions where the art demonstrates no distinguishing featuresof the polynucleotide/sequences of the present invention.

[0079] In specific embodiments, the polynucleotides of the invention areat least 15, at least 30, at least 50, at least 100, at least 125, atleast 500, or at least 1000 continuous nucleotides but are less than orequal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotidesof the invention comprise a portion of the coding sequences, asdisclosed herein, but do not comprise all or a portion of any intron. Inanother embodiment, the polynucleotides comprising coding sequences donot contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′to the gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

[0080] As used herein, a “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:1 or the cDNA containedwithin the clone deposited with the ATCC. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without a signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

[0081] In the present invention, the full length sequence identified asSEQ ID NO:1 was often generated by overlapping sequences contained inone or more clones (contig analysis). A representative clone containingall or most of the sequence for SEQ ID NO:1 was deposited with theAmerican Type Culture Collection (“ATCC”). As shown in Table I, eachclone is identified by a cDNA Clone ID (Identifier) and the ATCC DepositNumber. The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. The ATCC deposit was made pursuant to the terms of theBudapest Treaty on the international recognition of the deposit ofmicroorganisms for purposes of patent procedure. The deposited clone isinserted in the pSport1 plasmid (Life Technologies) using SalI and NotIrestriction sites described herein.

[0082] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined above. Therefore, as is known in the art for any DNA sequencedetermined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0083] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A-C (SEQ ID NO:1), a nucleic acid molecule of thepresent invention encoding the HLRRSI1 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A-C (SEQ ID NO:1) wasdiscovered in a human brain and testis cDNA library.

[0084] A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:1, the complementthereof, or the cDNA within the clone deposited with the ATCC.“Stringent hybridization conditions” refers to an overnight incubationat 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65 degree C.

[0085] Also contemplated are nucleic acid molecules that hybridize tothe polynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

[0086] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0087] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in thesequence listing), or to a complementary stretch of T (or U) residues,would not be included in the definition of “polynucleotide,” since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

[0088] The polynucleotide of the present invention can be composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

[0089] The polypeptide of the present invention can be composed of aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

[0090] “SEQ ID NO:1” refers to a polynucleotide sequence while “SEQ IDNO:2” refers to a polypeptide sequence, both sequences are identified byan integer specified in Table I.

[0091] “A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

[0092] The term “organism” as referred to herein is meant to encompassany organism referenced herein, though preferably to eukaryoticorgansisms, more preferably to mammals, and most preferably to humans.

[0093] The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that described by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N.Y. Acad. Sci., 7;766:279-81, (1995)).

[0094] The polynucleotide and polypeptides of the present invention areuseful as probes for the identification and isolation of full-lengthcDNAs and/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarrays.

[0095] In addition, polynucleotides and polypeptides of the presentinvention may comprise one, two, three, four, five, six, seven, eight,or more membrane domains.

[0096] Also, in preferred embodiments the present invention providesmethods for further refining the biological function of thepolynucleotides and/or polypeptides of the present invention.

[0097] Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

[0098] In preferred embodiments, the invention provides methods foridentifying the glycosylation sites inherent in the polynucleotides andpolypeptides of the invention, and the subsequent alteration, deletion,and/or addition of said sites for a number of desirable characteristicswhich include, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

[0099] In further preferred embodiments, methods are provided forevolving the polynucleotides and polypeptides of the present inventionusing molecular evolution techniques in an effort to create and identifynovel variants with desired structural, functional, and/or physicalcharacteristics.

[0100] The present invention further provides for other experimentalmethods and procedures currently available to derive functionalassignments. These procedures include but are not limited to spotting ofclones on arrays, micro-array technology, PCR based methods (e.g.,quantitative PCR), anti-sense methodology, gene knockout experiments,and other procedures that could use sequence information from clones tobuild a primer or a hybrid partner.

[0101] As used herein the terms “modulate” or “modulates” refer to anincrease or decrease in the amount, quality or effect of a particularactivity, DNA, RNA, or protein.

[0102] Polynucleotides and Polypeptides of the Invention

[0103] Features of the Polypeptide Encoded by Gene No:1

[0104] The polypeptide of this gene provided as SEQ ID NO:2 (FIGS.1A-C), encoded by the polynucleotide sequence according to SEQ ID NO:1(FIGS. 1A-C), and/or encoded by the polynucleotide contained within thedeposited clone, HLRRSI1 (also refered to as GPCR12#99, GPCR12#100,SILL1A and/or SILL1B), has significant homology at the nucleotide andamino acid level to a number of leucine-rich repeat containing proteins,which include, for example, the human caspase recruitment protein 7protein (caspase_recruitment_protein; Genbank Accession No:gi|10198209;SEQ ID NO:3); the human nucleotide binding site protein protein(Nucleotide_Binding_site; Genbank Accession No:gi|10198207; SEQ IDNO:4); and the human cryopyrin protein (cryopyrin; Genbank AccessionNo:gi|17027237; SEQ ID NO:33). An alignment of the HLRRSI1 polypeptidewith these proteins is provided in FIGS. 2A-C.

[0105] The determined nucleotide sequence of the HLRRSI1 cDNA in FIGS.1A-C (SEQ ID NO:1) contains an open reading frame encoding a protein ofabout 625 amino acid residues, with a deduced molecular weight of about68.9 kDa. The amino acid sequence of the predicted HLRRSI1 polypeptideis shown in FIGS. 1A-C (SEQ ID NO:2). The HLRRSI1 protein shown in FIGS.1A-C was determined to share significant identity and similarity toseveral known leucine-rich repeat containing proteins, particularly,caspase recruitment proteins. Specifically, the HLRRSI1 protein shown inFIGS. 1A-C was determined to be about 36.3% identical and 44.0% similarto the human caspase recruitment protein 7 protein(caspase_recruitment_protein; Genbank Accession No:gi|10198209; SEQ IDNO:3); to be about 35.0% identical and 42.2% similar to the humannucleotide binding site protein (Nucleotide_Binding_site; GenbankAccession No:gi|10198207; SEQ ID NO:4); and to be about 35.7% identicaland 46.0% similar the human cryopyrin protein (cryopyrin; GenbankAccession No:gi|7027237; SEQ ID NO:33) as shown in FIG. 5. Thenucleotide binding site protein presumably functions in apoptosis andinflammation [Bertin, 2000]. HLRRSI-1 is also 24% identical and 40%similar to the mouse protein MATER, a protein that is associated withautoimmune premature ovarian failure [Tong, 1999 #35].

[0106] The human caspase recruitment protein 7 protein(caspase_recruitment_protein; Genbank Accession No:gi|10198209; SEQ IDNO:3) is a leucine-rich repeat protein. It comprisies a pyrin domainwhich is a domain found primarily in proteins involved in apoptosis andinflammatory proteins.

[0107] The human cryopyrin protein (cryopyrin; Genbank AccessionNo:gi|17027237; SEQ ID NO:33) is a leucine-rich repeat protein thatcontains a pyrin domain. Mutations in the cryopyrin protein have beenassociated with the incidence of familial cold autoinflammatory syndrome(FCAS, MIM 120100), commonly known as familial cold urticaria (FCU),which is an autosomal-dominant systemic inflammatory diseasecharacterized by intermittent episodes of rash, arthralgia, fever andconjunctivitis after generalized exposure to cold. In addition,mutations in the cryopyrin protein were also associated with theincidence of Muckle-Wells syndrome (MWS; MIM 191900), which is anautosomal-dominant periodic fever syndrome with a similar phenotypeexcept that symptoms are not precipitated by cold exposure and thatsensorineural hearing loss is frequently also present. Additionally, thecryopyrin protein is also believed to play a role in the regulation ofinflammation and apoptosis.

[0108] Analysis of the HLRRSI1 polypeptide indicates that it contains aleucine-rich repeat domain n the carboxy terminus with an allowedcysteine substituting for asparagine at position 10 of the repeat and anadditional region of non-canonical leucines and cysteines on its aminoterminus flanking sequence. The protein contains two hydrophobic regionsof sufficient length to span the membrane, although three putativetransmembrane domains are present. The HLRRSI-1 polypeptide does notcontain a signal sequence at the amino terminus. HLRRSI-1 is expresseddramatically in the small intestine and may function in the regulationof inflammatory responses and apoptosis within this tissue.

[0109] The HLRRSI1 polypeptide was predicted to comprise threetransmembrane domain using the TMPRED program (K Hofmann, W Stoffel,Biol. Chem., 347:166, 1993). The predicted transmembrane domains of theHLRRSI1 polypeptide is located from about amino acid 38 to about aminoacid 61, from about amino acid 115 to about amino acid 131, and/or fromabout amino acid 151 to about amino acid 167 of SEQ ID NO:2 (FIGS.1A-C). In this context, the term “about” may be construed to mean 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/orC-terminus of the above referenced polypeptide.

[0110] In preferred embodiments, the following transmembrane domainpolypeptide is encompassed by the present invention:GARVLGGLLSKALLPTALLLVTTR (SEQ ID NO:8), LFALCFVPFVCWIVCTV (SEQ ID NO:9),and/or SVYLLFITSVLSSAPVA (SEQ ID NO:10). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 transmembrane domain polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0111] In preferred embodiments, the following N-terminal deletionmutants are encompassed by the present invention: G1-R24, A2-R24,R3-R24, V4-R24, L5-R24, G6-R24, G7-R24, L8-R24, L9-R24, S10-R24,K11-R24, A12-R24, L13-R24, L14-R24, P15-R24, T16-R24, A17-R24, and/orL18-R24 of SEQ ID NO:8. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 transmembrane domain N-terminal deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0112] In preferred embodiments, the following C-terminal deletionmutants are encompassed by the present invention: G1-R24, G1-T23,G1-T22, G1-V21, G1-L20, G1-L19, G1-L18, G1-A17, G1-T16, G1-P15, G1-L14,G1-L13, G1-A12, G1-K11, G1-S10, G1-L9, G1-L8, and/or G1-G7 of SEQ IDNO:8. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the HLRRSI1transmembrane domain C-terminal deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

[0113] In preferred embodiments, the following N-terminal deletionmutants are encompassed by the present invention: L1-V17, F2-V17,A3-V17, L4-V17, C5-V17, F6-V17, V7-V17, P8-V17, F9-V17, V10-V17, and/orC11-V17 of SEQ ID NO:9. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 transmembrane domain N-terminal deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0114] In preferred embodiments, the following C-terminal deletionmutants are encompassed by the present invention: L1-V17, L1-T16,L1-C15, L1-V14, L1-113, L1-W12, L1-C11, L1-V10, L1-F9, L1-P8, and/orL1-V7 of SEQ ID NO:9. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 transmembrane domain C-terminal deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0115] In preferred embodiments, the following N-terminal deletionmutants are encompassed by the present invention: S1-A17, V2-A17,Y3-A17, L4-A17, L5-A17, F6-A17, 17-A17, T8-A17, S9-A17, V10-A17, and/orL11-A17 of SEQ ID NO:10. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 transmembrane domain N-terminal deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0116] In preferred embodiments, the following C-terminal deletionmutants are encompassed by the present invention: S1-A17, S1-V16,S1-P15, S1-A14, S1-S13, S1-S12, S1-L11, S1-V10, S1-S9, S1-T8, and/orS1-17 of SEQ ID NO:10. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 transmembrane domain C-terminal deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0117] Based upon the strong homology to members of the leucine-richrepeat containing proteins, the HLRRSI1 polypeptide is expected to shareat least some biological activity with leucine-rich repeat containingproteins, preferably with members of the caspase recruitment proteinfamily of leucine-rich repeat containing proteins, particularly thecaspase recruitment protein family members referenced herein, and morepreferably with leucine-rich repeat containing proteins found withinbone marrow cells and tissues.

[0118] Alternatively, based upon the strong homology to members of theleucine-rich repeat containing proteins, the HLRRSI1 polypeptide isexpected to share at least some biological activity with humannucleotide binding site protein, and possibly with the mouse proteinMATER described herein.

[0119] Moreover, based upon the strong homology to members of theleucine-rich repeat containing proteins, the HLRRSI1 polypeptide isexpected to share at least some biological activity with pyrin domaincontaining proteins, particularly the pyrin domain containing proteinsreferenced herein.

[0120] The HLRRSI1 polypeptide was also determined to comprise severalconserved leucine residues, at amino acid 2, 9, 42, 45, 46, 51, 57, 68,115, 118, 132, 154, 176, 179, 182, 199, 247, 264, 284, 356, 359, 408,424, 444, 463, 485, 494, 496, 509, 537, 541, 547, 552, 557, 565, 569,576, 579, 581, 586, 594, and 604 of SEQ ID No: 2 (FIGS. 1A-C). Moreover,the HLRRSI1 polypeptide also was determined to comprise a fewdifferentially conserved leucine residues, at amino acid 8, 12, 56, 161,204, 219, 240, 243, 310, 406, 428, 466, 491, and 550 of SEQ ID No:2(FIGS. 1A-C). The conservation of leucines at key amino acid residues isconsistent with the HLRRSI1 polypeptide as being a member of theleucine-rich repeat containing protein family, and may be indicative ofconserved structural features, which may correlate with conservation ofprotein function and/or activity.

[0121] The HLRRSI1 polypeptide was also determined to comprise severalconserved cysteines, at amino acid 29, 129, 299, 358, 396, 461, 518, and574 of SEQ ID No: 2 (FIGS. 1A-C). Conservation of cysteines at key aminoacid residues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0122] Expression profiling designed to measure the steady state mRNAlevels encoding the HLRRSI1 polypeptide showed predominately highexpression levels in small intestine, and to a lesser extent, in liver,lymph node, and spleen tissue (See FIG. 4).

[0123] Consistent with the strong homology to caspase rescruitmentproteins, antisense assays have shown the HLRRSI1 polypeptide to beinvolved in the regulation of mammalian NF-κB and apoptosis pathways.Subjecting A549 cells with an effective amount of a pool of fiveantisense oligoncleotides (SEQ ID NO:34, 35, 36, 37, and 38) directedagainst the coding region of the HLRRSI polynucleotide resulted in asignificant increase in IκBα expression/activity providing convincingevidence that HLRRSI at least regulates the activity and/or expressionof IκBα either directly, or indirectly. Moreover, the results suggestthat HLRRSI is involved in the negative regulation of NF-κB/IκBαactivity and/or expression, either directly or indirectly. The IκBαassay used is described in Example 57 and was based upon the analysis ofIκBα activity as a downstream marker for proliferative signaltransduction events.

[0124] The upregulation of IkBa due to the downregulation of HLRRSI1places this leucine-rich repeat protein into a signalling pathwaypotentially involved in apoptotic events. This gives the opportunity toregulate downstream events via the activity of the protein HLRRSI1 withantisense polynucleotides, polypeptides or low molecular chemicals withthe potential of achieving a therapeutic effect in cancer, autoimmunediseases. In addition to cancer and immunological disorders, NF-kB hassignificant roles in other diseases (Baldwin, A. S., J. Clin Invest.107, :3-6 (2001)). NF-kB is a key factor in the pathophysiology ofischemia-reperfusion injury and heart failure (Valen, G., Yan. Z Q,Hansson, G K, J. Am. Coll. Cardiol. 38, 307-14 (2001)). Furthermore,NF-kB has been found to be activated in experimental renal disease(Guijarro C, Egido J., Kidney Int. 59, 415-425 (2001)). As HLRRSI1 ishighly expressed in small intestine there is potential involvement ofHLRRSI1 in treating gastrointenstinal diseases, particularly for cancersthrough the administration of HLRRSI1 and/or agonists thereof.

[0125] In preferred embodiments, HLRRSI1 polynucleotides andpolypeptides, including fragments thereof, are useful for treating,diagnosing, and/or ameliorating proliferative disorders, cancers,ischemia-reperfusion injury, heart failure, immuno compromisedconditions, HIV infection, and gastrointestinal diseases.

[0126] Moreover, HLRRSI1 polynucleotides and polypeptides, includingfragments thereof, are useful for increasing NF-kB activity, increasingapoptotic events, and/or decreasing IεBα expression or activity levels.

[0127] In preferred embodiments, antagonists directed against HLRRSI1are useful for treating, diagnosing, and/or ameliorating autoimmunedisorders, disorders related to hyper immune activity, inflammatoryconditions, disorders related to aberrant acute phase responses,hypercongenital conditions, birth defects, necrotic lesions, wounds,organ transplant rejection, conditions related to organ transplantrejection, disorders related to aberrant signal transduction,proliferating disorders, cancers, HIV, and HIV propagation in cellsinfected with other viruses.

[0128] Moreover, antagonists directed against HLRRSI1 are useful fordecreasing NF-kB activity, decreasing apoptotic events, and/orincreasing IκBα expression or activity levels.

[0129] In preferred embodiments, agonists directed against HLRRSI1 areuseful for treating, diagnosing, and/or ameliorating autoimmunediorders, disorders related to hyper immune activity, hypercongenitalconditions, birth defects, necrotic lesions, wounds, disorders relatedto aberrant signal transduction, immuno compromised conditions, HIVinfection, proliferating disorders, and/or cancers.

[0130] Moreover, agonists directed against HLRRSI1 are useful forincreasing NF-kB activity, increasing apoptotic events, and/ordecreasing IκBα expression or activity levels, particularly ingastrointestinal tissue such as the small intestine, for example.

[0131] The HLRRSI1 polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude detecting, prognosing, treating, preventing, and/or amelioratingthe following diseases and/or disorders, disorders related to aberrantapoptosis regulation, disorders related to aberrant cell adhesionregulation, and disorders related to aberrant cellular proliferation,for example, in addition to, immune, and hepatic disorders.

[0132] The HLRRSI1 polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating signal transduction activity, in various cells,tissues, and organisms, and particularly in mammalian small intestine,liver, lymph node, and spleen tissue, preferably human tissue.

[0133] The strong homology to human leucine-rich repeat containingproteins, combined with the predominate localized expression in smallintestine and its involvement in IkB modulation, suggests the HLRRSI1polynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing gastrointesinal diseases and/or disorders,which include, but are not limited to, ulcers, irritable bowel syndrome,diarrhea, polyps, absorption disorders, constipation, diverticulitis,vascular disease of the intestines, intestinal obstruction, intestinalinfections, clcerative colitis, Crohn's Disease, hereditaryhemochromatosis, gastroenteritis, mesenteric ischemia, mesentericinfarction, in addition to, metabolic diseases and/or disorders.

[0134] The strong homology to human leucine-rich repeat containingproteins, combined with the expression in lymph node and spleen tissueand its involvement in IkB modulation, suggests the HLRRSI1polynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing immune and hematopoietic diseases and/ordisorders, which include, but are not limited to, anemia, pancytopenia,leukopenia, thrombocytopenia or leukemia since stromal cells areimportant in the production of cells of hematopoietic lineages.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, the Examples, and elsewhere herein.Briefly, the uses include bone marrow cell ex-vivo culture, bone marrowtransplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia. The gene product may also be involved inlymphopoiesis, therefore, it can be used in immune disorders such asinfection, inflammation, allergy, immunodeficiency etc. In addition,this gene product may have commercial utility in the expansion of stemcells and committed progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types.

[0135] Moreover, the HLRRSI1 polypeptide may be useful for modulatingcytokine production, antigen presentation, or other processes, such asfor boosting immune responses, etc. Expression in cells of lymphoidorigin, indicates the natural gene product would be involved in immunefunctions. Therefore it would also be useful as an agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, astissuemarkers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0136] The HLRRSI1 polynucleotides and polypeptides, including agonists,antagonists, and/or fragments thereof, though preferably agonists of thepresent invention have uses which include, for example, modulatingcellular proliferation. Likewise, the HLRRSI1 polynucleotides andpolypeptides, including agonists, antagonists, and/or fragments thereof,though preferably agonisits of the present invention, may be useful forthe treatment, detection, amelioration, and/or prevention of disordersrelated, or directly linked to, aberrant cellular proliferation, suchas, for example, cancers.

[0137] Moreover, HLRRSI1 polynucleotides and polypeptides, includingfragments and agonists thereof, may have uses which include treating,diagnosing, prognosing, and/or preventing hyperproliferative disorders,particularly of the immune, hematopoietic, pulmonary, and reproductivesystems. Such disorders may include, for example, cancers, andmetastasis.

[0138] The HLRRSI1 polynucleotides and polypeptides, including fragmentsand agonists thereof, may have uses which include, either directly orindirectly, for boosting immune responses.

[0139] The HLRRSI1 polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, may have uses which include identificationof modulators of HLRRSI1 function including antibodies (for detection orneutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains of the HLRRSI1 protein could be usedas diagnostic agents of hematopoietic and inflammatory conditions inpatients, are useful in monitoring the activation of signal transductionpathways, and can be used as a biomarker for the involvement ofleucine-rich repeat containing proteins in disease states, and in theevaluation of inhibitors of leucine-rich repeat containing proteins invivo.

[0140] HLRRSI1 polypeptides and polynucleotides have additional useswhich include diagnosing diseases related to the over and/or underexpression of HLRRSI1 by identifying mutations in the HLRRSI1 gene byusing HLRRSI1 sequences as probes or by determining HLRRSI1 protein ormRNA expression levels. HLRRSI1 polypeptides may be useful for screeningcompounds that affect the activity of the protein. HLRRSI1 peptides canalso be used for the generation of specific antibodies and as bait inyeast two hybrid screens to find proteins the specifically interact withHLRRSI1 (described elsewhere herein).

[0141] Recently, leucine-rich repeat containing proteins have beendirectly implicated in the pathogenesis of Bernard-Soulier syndrome(BSS), a hereditary qualitative platelet disorder. The disorder has beenlinked to the qualitative or quantitative abnormality of the plateletglycoprotein (GP) Ib/IX/V complex, which is formed by the aggregation ofseveral leucine-rich repeat containing proteins (Hayashi, T.; Suzuki,K., Semin. Thromb. Hemost., 26(1): 53-9 (2000).

[0142] In preferred embodiments, HLRRSI1 polypeptides, includingantagonists, and fragments thereof, have uses which include, forexample, the treatment, detection, prevention, prognosis, and/oramelioration of platelet disorders, including, but not limited to,Bernard-Soulier syndrome (BSS).

[0143] As descussed elsewhere herein, the Drosophia Toll proteins,including the human homologues thereof, have been implicated inmodulating development and in non-infectious disease (Schuster, J M.,Nelson, P S, J. Leukoc, Biol., 67(6):767-73, (2000)).

[0144] In preferred embodiments, HLRRSI1 polypeptides, includingantagonists, and fragments thereof, have uses which include, forexample, the treatment, detection, prevention, prognosis, and/oramelioration of developmental disorders, and non-infectious disorders,such as innate immunity to bacterial pathogens, adaptive immuneresponses, and others as listed in Schuster, J M et al., supra.

[0145] Leucine-rich repeat proteins have also been implicated in theincidence of systemic lupus erythematosus (Koarada, S., Tada, Y.,Ushiyama, O., Morito, F., Suzuki, N., Ohta, A., Miyake, K., Kimoto, M.,Nagasawa, K, Arthritis, Rheum., 42(12):2593-600, (1999)).

[0146] In preferred embodiments, HLRRSI1 polypeptides, includingantagonists, and fragments thereof, have uses which include, forexample, the treatment, detection, prevention, prognosis, and/oramelioration of immune disorders, which include, for example, systemiclupus erythematosus.

[0147] As described elsewhere herein, leucine-rich repeat proteins arealso implicated in a number of processes related to pathogen and/ordisease resistance in plants. This is of particular significance, sincemammals, including, humans, have homologues of some of these proteinswhich are thought to function in innate immune responses ((Dixon, M S.,Hatzixanthis, K., Jones, DA., Harrison, K., Jones, JD, Plant, Cell.,10(11):1915-25, (1998); Ellis, J., Jones, D, Curr, Opin, Plant, Biol.,1(4):288-93, (1998); Collins, N., Drake, J., Ayliffe, M., Sun, Q.,Ellis, J., Hulbert, S., Pryor, T, Plant, Cell., 11(7):1365-76, (1999);Wang, Z X., Yano, M., Yamanouchi, U., Iwamoto, M., Monna, L., Hayasaka,H., Katayose, Y., Sasaki, T, Plant, J., 19(1):55-64, (1999); Richter,TE., Ronald, PC, Plant, Mol, Biol., 42(1):195-204, (2000); Graham, M A.,k, L F., Lohnes, D., Cregan, P., Shoemaker, R C, Genome., 43(1):86-93,(2000); and He, Z., Wang, Z Y., Li, J., Zhu, Q., Lamb, C., Ronald, P.,Chory, J. Science. 30., 288(5475):2360-3, (2000)).

[0148] Moreover, the HLRRSI1 polypeptide shares significant homolgy withcaspase recruitment proteins. Aberrations of such proteins have beenimplicated in the incidence of a number of disorders related to aberrantapoptosis regulation, and in various inflammatory disorders(Srinivasula, S M., Ahmad, M., Lin, J H., Poyet, J L., Fernandes,Alnenri, T., Tsichlis, P N., Alnemri, E S, J. Biol, Chem. 18.,274(25):17946-54, (1999)).

[0149] Although it is believed the encoded polypeptide may share atleast some biological activities with leucine-rich repeat containingproteins (particularly caspase recruitment proteins), a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the HLRRSI1 polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied. For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from diseased small intestine tissue, as compared to, normaltissue might indicate a function in modulating immune and/orhematopoietic function, for example. In the case of HLRRSI1, smallintestine, liver, lymph node, and spleen tissue should be used, forexample, to extract RNA to prepare the probe.

[0150] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the HLRRSI1 gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiments. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. In the case of HLRRSI1, a disease correlationrelated to HLRRSI1 may be made by comparing the mRNA expression level ofHLRRSI1 in normal tissue, as compared to diseased tissue (particularlydiseased tissue isolated from the following: small intestine, liver,lymph node, and spleen tissue). Significantly higher or lower levels ofHLRRSI1 expression in the diseased tissue may suggest HLRRSI1 plays arole in disease progression, and antagonists against HLRRSI1polypeptides would be useful therapeutically in treating, preventing,and/or ameliorating the disease. Alternatively, significantly higher orlower levels of HLRRSI1 expression in the diseased tissue may suggestHLRRSI1 plays a defensive role against disease progression, and agonistsof HLRRSI1 polypeptides may be useful therapeutically in treating,preventing, and/or ameliorating the disease. Also encompassed by thepresent invention are quantitative PCR probes corresponding to thepolynucleotide sequence provided as SEQ ID NO:1 (FIGS. 1A-C).

[0151] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theHLRRSI1, transforming yeast deficient in leucine-rich repeat containingprotein activity, preferably caspase recruitment protein activity, forexample, and assessing their ability to grow would provide convincingevidence the HLRRSI1 polypeptide has leucine-rich repeat containingprotein activity, and potentially caspase recruitment protein activity.Additional assay conditions and methods that may be used in assessingthe function of the polynucleotides and polypeptides of the presentinvention are known in the art, some of which are disclosed elsewhereherein.

[0152] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype. Such knock-outexperiments are known in the art, some of which are disclosed elsewhereherein.

[0153] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a small intestine,liver, lymph node, and spleen tissue specific promoter), or it can beexpressed at a specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0154] In the case of HLRRSI1 transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (gastrointestinal, hepatic, immune, hematopoieticdisorders, in addition to cancers, etc.) may lead to understanding thefunction of the gene. Therefore, the application of antisense and/orsense methodology to the creation of transgenic mice or rats to refinethe biological function of the polypeptide is encompassed by the presentinvention.

[0155] In preferred embodiments, the following N-terminal deletionmutants are encompassed by the present invention: M1-F625, L2-F625,A3-F625, Q4-F625, P5-F625, Q6-F625, R7-F625, L8-F625, L9-F625, F10-F625,111-F625, L12-F625, D13-F625, G14-F625, A15-F625, D16-F625, E17-F625,L18-F625, P19-F625, A20-F625, L21-F625, G22-F625, G23-F625, P24-F625,E25-F625, A26-F625, A27-F625, P28-F625, C29-F625, T30-F625, D31-F625,P32-F625, F33-F625, E34-F625, A35-F625, A36-F625, S37-F625, G38-F625,A39-F625, R40-F625, V41-F625, L42-F625, G43-F625, G44-F625, L45-F625,L46-F625, S47-F625, K48-F625, A49-F625, L50-F625, L51-F625, P52-F625,T53-F625, A54-F625, L55-F625, L56-F625, L57-F625, V58-F625, T59-F625,T60-F625, R61-F625, A62-F625, A63-F625, A64-F625, P65-F625, G66-F625,R67-F625, L68-F625, Q69-F625, G70-F625, R71-F625, L72-F625, C73-F625,S74-F625, P75-F625, Q76-F625, C77-F625, A78-F625, E79-F625, V80-F625,R81-F625, G82-F625, F83-F625, S84-F625, D85-F625, K86-F625, D87-F625,K88-F625, K89-F625, K90-F625, Y91-F625, F92-F625, Y93-F625, K94-F625,F95-F625, F96-F625, R97-F625, D98-F625, E99-F625, R100-F625, R101-F625,A102-F625, E103-F625, R104-F625, A105-F625, Y106-F625, R107-F625,F108-F625, V109-F625, K110-F625, E111-F625, N112-F625, E113-F625,T114-F625, L115-F625, F116-F625, A117-F625, L118-F625, C119-F625,F120-F625, V121-F625, P122-F625, F123-F625, V124-F625, C125-F625,W126-F625, 1127-F625, V128-F625, C129-F625, T130-F625, V131-F625,L132-F625, R133-F625, Q134-F625, Q135-F625, L136-F625, E137-F625,L138-F625, G139-F625, R140-F625, D141-F625, L142-F625, S143-F625,R144-F625, T145-F625, S146-F625, K147-F625, T148-F625, T149-F625,T150-F625, S151-F625, V152-F625, Y153-F625, L154-F625, L155-F625,F156-F625, 1157-F625, T158-F625, S159-F625, V160-F625, L161-F625,S162-F625, S163-F625, A164-F625, P165-F625, V166-F625, A167-F625,D168-F625, G169-F625, P170-F625, R171-F625, L172-F625, Q173-F625,G174-F625, D175-F625, L176-F625, R177-F625, N178-F625, L179-F625,C180-F625, R181-F625, L182-F625, A183-F625, R184-F625, E185-F625,G186-F625, V187-F625, L188-F625, G189-F625, R190-F625, R191-F625,A192-F625, Q193-F625, F194-F625, A195-F625, E196-F625, K197-F625,E198-F625, L199-F625, E200-F625, Q201-F625, L202-F625, E203-F625,L204-F625, R205-F625, G206-F625, S207-F625, K208-F625, V209-F625,Q210-F625, T211-F625, L212-F625, F213-F625, L214-F625, S215-F625,K216-F625, K217-F625, E218-F625, L219-F625, P220-F625, G221-F625,V222-F625, L223-F625, E224-F625, T225-F625, E226-F625, V227-F625,T228-F625, Y229-F625, Q230-F625, F231-F625, 1232-F625, D233-F625,Q234-F625, S235-F625, F236-F625, Q237-F625, E238-F625, F239-F625,L240-F625, A241-F625, A242-F625, L243-F625, S244-F625, Y245-F625,L246-F625, L247-F625, E248-F625, D249-F625, G250-F625, G251-F625,V252-F625, P253-F625, R254-F625, T255-F625, A256-F625, A257-F625,G258-F625, G259-F625, V260-F625, G261-F625, T262-F625, L263-F625,L264-F625, R265-F625, G266-F625, D267-F625, A268-F625, Q269-F625,P270-F625, H271-F625, S272-F625, H273-F625, L274-F625, V275-F625,L276-F625, T277-F625, T278-F625, R279-F625, F280-F625, L281-F625,F282-F625, G283-F625, L284-F625, L285-F625, S286-F625, A287-F625,E288-F625, R289-F625, M290-F625, R291-F625, D292-F625, 1293-F625,E294-F625, R295-F625, H296-F625, F297-F625, G298-F625, C299-F625,M300-F625, V301-F625, S302-F625, E303-F625, R304-F625, V305-F625,K306-F625, Q307-F625, E308-F625, A309-F625, L310-F625, R311-F625,W312-F625, V313-F625, Q314-F625, G315-F625, Q316-F625, G317-F625,Q318-F625, G319-F625, C320-F625, P321-F625, G322-F625, V323-F625,A324-F625, P325-F625, E326-F625, V327-F625, T328-F625, E329-F625,G330-F625, A331-F625, K332-F625, G333-F625, L334-F625, E335-F625,D336-F625, T337-F625, E338-F625, E339-F625, P340-F625, E341-F625,E342-F625, E343-F625, E344-F625, E345-F625, G346-F625, E347-F625,E348-F625, P349-F625, N350-F625, Y351-F625, P352-F625, L353-F625,E354-F625, L355-F625, L356-F625, Y357-F625, C358-F625, L359-F625,Y360-F625, E361-F625, T362-F625, Q363-F625, E364-F625, D365-F625,A366-F625, F367-F625, V368-F625, R369-F625, Q370-F625, A371-F625,L372-F625, C373-F625, R374-F625, F375-F625, P376-F625, E377-F625,L378-F625, A379-F625, L380-F625, Q381-F625, R382-F625, V383-F625,R384-F625, F385-F625, C386-F625, R387-F625, M388-F625, D389-F625,V390-F625, A391-F625, V392-F625, L393-F625, S394-F625, Y395-F625,C396-F625, V397-F625, R398-F625, C399-F625, C400-F625, P401-F625,A402-F625, G403-F625, Q404-F625, A405-F625, L406-F625, R407-F625,L408-F625, 1409-F625, S410-F625, C411-F625, R412-F625, L413-F625,V414-F625, A415-F625, A416-F625, Q417-F625, E418-F625, K419-F625,K420-F625, K421-F625, K422-F625, S423-F625, L424-F625, G425-F625,K426-F625, R427-F625, L428-F625, Q429-F625, A430-F625, S431-F625,L432-F625, G433-F625, G434-F625, G435-F625, S436-F625, S437-F625,Q438-F625, G439-F625, T440-F625, T441-F625, K442-F625, Q443-F625,L444-F625, P445-F625, A446-F625, S447-F625, L448-F625, L449-F625,H450-F625, P451-F625, L452-F625, F453-F625, Q454-F625, A455-F625,M456-F625, T457-F625, D458-F625, P459-F625, L460-F625, C461-F625,H462-F625, L463-F625, S464-F625, S465-F625, L466-F625, T467-F625,L468-F625, S469-F625, H470-F625, C471-F625, K472-F625, L473-F625,P474-F625, D475-F625, A476-F625, V477-F625, C478-F625, R479-F625,D480-F625, L481-F625, S482-F625, E483-F625, A484-F625, L485-F625,R486-F625, A487-F625, A488-F625, P489-F625, A490-F625, L491-F625,T492-F625, E493-F625, L494-F625, G495-F625, L496-F625, L497-F625,H498-F625, N499-F625, R500-F625, L501-F625, S502-F625, E503-F625,A504-F625, G505-F625, L506-F625, R507-F625, M508-F625, L509-F625,S510-F625, E511-F625, G512-F625, L513-F625, A514-F625, W515-F625,P516-F625, Q517-F625, C518-F625, R519-F625, V520-F625, Q521-F625,T522-F625, V523-F625, R524-F625, V525-F625, Q526-F625, L527-F625,P528-F625, D529-F625, P530-F625, Q531-F625, R532-F625, G533-F625,L534-F625, Q535-F625, Y536-F625, L537-F625, V538-F625, G539-F625,M540-F625, L541-F625, R542-F625, Q543-F625, S544-F625, P545-F625,A546-F625, L547-F625, T548-F625, T549-F625, L550-F625, D551-F625,L552-F625, S553-F625, G554-F625, C555-F625, Q556-F625, L557-F625,P558-F625, A559-F625, P560-F625, M561-F625, V562-F625, T563-F625,Y564-F625, L565-F625, C566-F625, A567-F625, V568-F625, L569-F625,Q570-F625, H571-F625, Q572-F625, G573-F625, C574-F625, G575-F625,L576-F625, Q577-F625, T578-F625, L579-F625, S580-F625, L581-F625,A582-F625, S583-F625, V584-F625, E585-F625, L586-F625, S587-F625,E588-F625, Q589-F625, S590-F625, L591-F625, Q592-F625, E593-F625,L594-F625, Q595-F625, A596-F625, V597-F625, K598-F625, R599-F625,A600-F625, K601-F625, P602-F625, D603-F625, L604-F625, V605-F625,1606-F625, T607-F625, H608-F625, P609-F625, A610-F625, L611-F625,D612-F625, G613-F625, H614-F625, P615-F625, Q616-F625, P617-F625,P618-F625, and/or K619-F625 of SEQ ID NO:2. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of the HLRRSI1 N-terminal deletion polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

[0156] In preferred embodiments, the following C-terminal deletionmutants are encompassed by the present invention: M1-F625, M1-T624,M1-S623, M1-1622, M1-L621, M1-E620, M1-K619, M1-P618, M1-P617, M1-Q616,M1-P615, M1-H614, M1-G613, M1-D612, M1-L611, M1-A610, M1-P609, M1-H608,M1-T607, M1-1606, M1-V605, M1-L604, M1-D603, M1-P602, M1-K601, M1-A600,M1-R599, M1-K598, M1-V597, M1-A596, M1-Q595, M1-L594, M1-E593, M1-Q592,M1-L591, M1-S590, M1-Q589, M1-E588, M1-S587, M1-L586, M1-E585, M1-V584,M1-S583, M1-A582, M1-L581, M1-S580, M1-L579, M1-T578, M1-Q577, M1-L576,M1-G575, M1-C574, M1-G573, M1-Q572, M1-H571, M1-Q570, M1-L569, M1-V568,M1-A567, M1-C566, M1-L565, M1-Y564, M1-T563, M1-V562, M1-M561, M1-P560,M1-A559, M1-P558, M1-L557, M1-Q556, M1-C555, M1-G554, M1-S553, M1-L552,M1-D551, M1-L550, M1-T549, M1-T548, M1-L547, M1-A546, M1-P545, M1-S544,M1-Q543, M1-R542, M1-L541, M1-M540, M1-G539, M1-V538, M1-L537, M1-Y536,M1-Q535, M1-L534, M1-G533, M1-R532, M1-Q531, M1-P530, M1-D529, M1-P528,M1-L527, M1-Q526, M1-V525, M1-R524, M1-V523, M1-T522, M1-Q521, M1-V520,M1-R519, M1-C518, M1-Q517, M1-P516, M1-W515, M1-A514, M1-L513, M1-G512,M1-E511, M1-S510, M1-L509, M1-M508, M1-R507, M1-L506, M1-G505, M1-A504,M1-E503, M1-S502, M1-L501, M1-R500, M1-N499, M1-H498, M1-L497, M1-L496,M1-G495, M1-L494, M1-E493, M1-T492, M1-L491, M1-A490, M1-P489, M1-A488,M1-A487, M1-R486, M1-L485, M1-A484, M1-E483, M1-S482, M1-L481, M1-D480,M1-R479, M1-C478, M1-V477, M1-A476, M1-D475, M1-P474, M1-L473, M1-K472,M1-C471, M1-H470, M1-S469, M1-L468, M1-T467, M1-L466, M1-S465, M1-S464,M1-L463, M1-H462, M1-C461, M1-L460, M1-P459, M1-D458, M1-T457, M1-M456,M1-A455, M1-Q454, M1-F453, M1-L452, M1-P451, M1-H450, M1-L449, M1-L448,M1-S447, M1-A446, M1-P445, M1-L444, M1-Q443, M1-K442, M1-T441, M1-T440,M1-G439, M1-Q438, M1-S437, M1-S436, M1-G435, M1-G434, M1-G433, M1-L432,M1-S431, M1-A430, M1-Q429, M1-L428, M1-R427, M1-K426, M1-G425, M1-L424,M1-S423, M1-K422, M1-K421, M1-K420, M1-K419, M1-E418, M1-Q417, M1-A416,M1-A415, M1-V414, M1-L413, M1-R412, M1-C411, M1-S410, M1-1409, M1-L408,M1-R407, M1-L406, M1-A405, M1-Q404, M1-G403, M1-A402, M1-P401, M1-C400,M1-C399, M1-R398, M1-V397, M1-C396, M1-Y395, M1-S394, M1-L393, M1-V392,M1-A391, M1-V390, M1-D389, M1-M388, M1-R387, M1-C386, M1-F385, M1-R384,M1-V383, M1-R382, M1-Q381, M1-L380, M1-A379, M1-L378, M1-E377, M1-P376,M1-F375, M1-R374, M1-C373, M1-L372, M1-A371, M1-Q370, M1-R369, M1-V368,M1-F367, M1-A366, M1-D365, M1-E364, M1-Q363, M1-T362, M1-E361, M1-Y360,M1-L359, M1-C358, M1-Y357, M1-L356, M1-L355, M1-E354, M1-L353, M1-P352,M1-Y351, M1-N350, M1-P349, M1-E348, M1-E347, M1-G346, M1-E345, M1-E344,M1-E343, M1-E342, M1-E341, M1-P340, M1-E339, M1-E338, M1-T337, M1-D336,M1-E335, M1-L334, M1-G333, M1-K332, M1-A331, M1-G330, M1-E329, M1-T328,M1-V327, M1-E326, M1-P325, M1-A324, M1-V323, M1-G322, M1-P321, M1-C320,M1-G319, M1-Q318, M1-G317, M1-Q316, M1-G315, M1-Q314, M1-V313, M1-W312,M1-R311, M1-L310, M1-A309, M1-E308, M1-Q307, M1-K306, M1-V305, M1-R304,M1-E303, M1-S302, M1-V301, M1-M300, M1-C299, M1-G298, M1-F297, M1-H296,M1-R295, M1-E294, M1-1293, M1-D292, M1-R291, M1-M290, M1-R289, M1-E288,M1-A287, M1-S286, M1-L285, M1-L284, M1-G283, M1-F282, M1-L281, M1-F280,M1-R279, M1-T278, M1-T277, M1-L276, M1-V275, M1-L274, M1-H273, M1-S272,M1-H271, M1-P270, M1-Q269, M1-A268, M1-D267, M1-G266, M1-R265, M1-L264,M1-L263, M1-T262, M1-G261, M1-V260, M1-G259, M1-G258, M1-A257, M1-A256,M1-T255, M1-R254, M1-P253, M1-V252, M1-G251, M1-G250, M1-D249, M1-E248,M1-L247, M1-L246, M1-Y245, M1-S244, M1-L243, M1-A242, M1-A241, M1-L240,M1-F239, M1-E238, M1-Q237, M1-F236, M1-S235, M1-Q234, M1-D233, M1-1232,M1-F231, M1-Q230, M1-Y229, M1-T228, M1-V227, M1-E226, M1-T225, M1-E224,M1-L223, M1-V222, M1-G221, M1-P220, M1-L219, M1-E218, M1-K217, M1-K216,M1-S215, M1-L214, M1-F213, M1-L212, M1-T211, M1-Q210, M1-V209, M1-K208,M1-S207, M1-G206, M1-R205, M1-L204, M1-E203, M1-L202, M1-Q201, M1-E200,M1-L199, M1-E198, M1-K197, M1-E196, M1-A195, M1-F194, M1-Q193, M1-A192,M1-R191, M1-R190, M1-G189, M1-L188, M1-V187, M1-G186, M1-E185, M1-R184,M1-A183, M1-L182, M1-R181, M1-C180, M1-L179, M1-N178, M1-R177, M1-L176,M1-D175, M1-G174, M1-Q173, M1-L172, M1-R171, M1-P170, M1-G169, M1-D168,M1-A167, M1-V166, M1-P165, M1-A164, M1-S163, M1-S162, M1-L161, M1-V160,M1-S159, M1-T158, M1-1157, M1-F156, M1-L155, M1-L154, M1-Y153, M1-V152,M1-S151, M1-T150, M1-T149, M1-T148, M1-K147, M1-S146, M1-T145, M1-R144,M1-S143, M1-L142, M1-D141, M1-R140, M1-G139, M1-L138, M1-E137, M1-L136,M1-Q135, M1-Q134, M1-R133, M1-L132, M1-V131, M1-T130, M1-C129, M1-V128,M1-1127, M1-W126, M1-C125, M1-V124, M1-F123, M1-P122, M1-V121, M1-F120,M1-C119, M1-L118, M1-A117, M1-F116, M1-L115, M1-T114, M1-E113, M1-N112,M1-E111, M1-K110, M1-V109, M1-F108, M1-R107, M1-Y106, M1-A05, M1-R104,M1-E103, M1-A102, M1-R101, M1-R100, M1-E99, M1-D98, M1-R97, M1-F96,M1-F95, M1-K94, M1-Y93, M1-F92, M1-Y91, M1-K90, M1-K89, M1-K88, M1-D87,M1-K86, M1-D85, M1-S84, M1-F83, M1-G82, M1-R81, M1-V80, M1-E79, M1-A78,M1-C77, M1-Q76, M1-P75, M1-S74, M1-C73, M1-L72, M1-R71, M1-G70, M1-Q69,M1-L68, M1-R67, M1-G66, M1-P65, M1-A64, M1-A63, M1-A62, M1-R61, M1-T60,M1-T59, M1-V58, M1-L57, M1-L56, M1-L55, M1-A54, M1-T53, M1-P52, M1-L51,M1-L50, M1-A49, M1-K48, M1-S47, M1-L46, M1-L45, M1-G44, M1-G43, M1-L42,M1-V41, M1-R40, M1-A39, M1-G38, M1-S37, M1-A36, M1-A35, M1-E34, M1-F33,M1-P32, M1-D31, M1-T30, M1-C29, M1-P28, M1-A27, M1-A26, M1-E25, M1-P24,M1-G23, M1-G22, M1-L21, M1-A20, M1-P19, M1-L18, M1-E17, M1-D16, M1-A15,M1-G14, M1-D13, M1-L12, M1-I11, M1-F10, M1-L9, M1-L8, and/or M1-R7 ofSEQ ID NO:2. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theHLRRSI1 C-terminal deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0157] The present invention also encompasses immunogenic and/orantigenic epitopes of the HLRRSI1 polypeptide.

[0158] In preferred embodiments, the following immunogenic and/orantigenic epitope polypeptides are encompassed by the present invention:amino acid residues from about amino acid 38 to about amino acid 61,from about amino acid 38 to about amino acid 46, from about amino acid47 to about amino acid 55, from about amino acid 53 to about amino acid61, from about amino acid 115 to about amino acid 131, from about aminoacid 115 to about amino acid 123, from about amino acid 124 to aboutamino acid 131, from about amino acid 151 to about amino acid 167, fromabout amino acid 151 to about amino acid 159, from about amino acid 159to about amino acid 167 of SEQ ID NO:2 (FIGS. 1A-C). In this context,the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids beyond the N-terminus and/or C-terminus of the abovereferenced polypeptides. Polynucleotides encoding these polypeptides arealso provided.

[0159] The HLRRSI1 polypeptides of the present invention were determinedto comprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the HLRRSI1 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the HLRRSI1 polypeptide toassociate with other polypeptides, particularly cognate ligand forHLRRSI1, or its ability to modulate certain cellular signal pathways.

[0160] The HLRRSI1 polypeptide was predicted to comprise nine PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem . . .260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0161] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: ALLLVTTRAAAPG(SEQ ID NO:16), EVRGFSDKDKKKY (SEQ ID NO:17), RDLSRTSKTTTSV (SEQ IDNO:18), QTLFLSKKELPGV (SEQ ID NO:19), SHLVLTTRFLFGL (SEQ ID NO:20),FGCMVSERVKQEA (SEQ ID NO:21), ALRLISCRLVAAQ (SEQ ID NO:22),GSSQGTTKQLPAS (SEQ ID NO:23), and/or QCRVQTVRVQLPD (SEQ ID NO:24).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of the HLRRSI1 PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0162] Specifically, the HLRRSI1 polypeptide was predicted to compriseone tyrosine phosphorylation site using the Motif algorithm (GeneticsComputer Group, Inc.). Such sites are phosphorylated at the tyrosineamino acid residue. The consensus pattern for tyrosine phosphorylationsites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y,where Y represents the phosphorylation site and ‘x’ represents anintervening amino acid residue. Additional information specific totyrosine phosphorylation sites can be found in Patschinsky T., HunterT., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci.U.S.A. 79:973-977(1982); Hunter T., J. Biol. Chem . . .257:4843-4848(1982), and Cooper J. A., Esch F. S., Taylor S. S., HunterT., J. Biol. Chem . . . 259:7835-7841(1984), which are herebyincorporated herein by reference.

[0163] In preferred embodiments, the following tyrosine phosphorylationsite polypeptides are encompassed by the present invention:FFRDERRAERAYRFVKE (SEQ ID NO:14). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HLRRSI1 PKC phosphorylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0164] The HLRRSI1 polypeptide has been shown to comprise oneglycosylation site according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0165] Asparagine phosphorylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem . .. 265:11397-11404(1990).

[0166] In preferred embodiments, the following asparagine glycosylationsite polypeptide is encompassed by the present invention: RFVKENETLFALCF(SEQ ID NO:14). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the HLRRSI1asparagine glycosylation site polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0167] The present invention encompasses the identification of compoundsand drugs which stimulate HLRRSI1 on the one hand (i.e., agonists) andwhich inhibit the function of HLRRSI1 on the other hand (i.e.,antagonists). In general, such screening procedures involve providingappropriate cells which express the polypeptide of the presentinvention. Such cells may include, for example, cells from mammals,yeast, Drosophila or E. coli. In a preferred embodiment, apolynucleotide encoding the polypeptide of the present invention may beemployed to transfect cells to thereby express the HLRRSI1 polypeptide.The expressed protein may then be contacted with a test compound toobserve binding, stimulation or inhibition of a functional response.

[0168] In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of HLRRSI1. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 78thru 1949 of SEQ ID NO:1, and the polypeptide corresponding to aminoacids 2 thru 625 of SEQ ID NO:2. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

[0169] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 2675 ofSEQ ID NO:1, b is an integer between 15 to 2689, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b is greater than or equal to a+14. TABLE I ATCC NT Total 5′NT Deposit SEQ NT Seq of Start 3′ NT Gene CDNA No. Z and ID. of Codon ofAA Seq Total AA of No. CloneID Date Vector No. X Clone of ORF ORF ID No.Y ORF 1. HLRRSI1- PTA-2679 PSport1 1 2689 75 1949 2 625 (also known asNov. 15, 2000 GPCR12#99, PTA-2674 GPCR12#100, Nov. 15, 2000 SILL1Aand/or SILL1B)

[0170] Table I summarizes the information corresponding to each “GeneNo.” described above. The nucleotide sequence identified as “NT SEQ IDNO:1” was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table I and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:1.

[0171] The cDNA Clone ID was deposited on the date and given thecorresponding deposit number listed in “ATCC Deposit No:Z and Date.”“Vector” refers to the type of vector contained in the cDNA Clone ID.

[0172] “Total NT Seq. Of Clone” refers to the total number ofnucleotides in the clone contig identified by “Gene No.” The depositedclone may contain all or most of the sequence of SEQ ID NO:1. Thenucleotide position of SEQ ID NO:1 of the putative start codon(methionine) is identified as “5′ NT of Start Codon of ORF.”

[0173] The translated amino acid sequence, beginning with themethionine, is identified as “AA SEQ ID NO:2,” although other readingframes can also be easily translated using known molecular biologytechniques. The polypeptides produced by these alternative open readingframes are specifically contemplated by the present invention.

[0174] The total number of amino acids within the open reading frame ofSEQ ID NO:2 is identified as “Total AA of ORF”.

[0175] SEQ ID NO:1 (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:2 (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:1 is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:1or the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:2 may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table I.

[0176] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0177] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and the predicted translated amino acid sequence identified as SEQID NO:2, but also a sample of plasmid DNA containing a cDNA of theinvention deposited with the ATCC, as set forth in Table I. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedcDNA, collecting the protein, and determining its sequence.

[0178] The present invention also relates to the genes corresponding toSEQ ID NO:1, SEQ ID NO:2, or the deposited clone. The corresponding genecan be isolated in accordance with known methods using the sequenceinformation disclosed herein. Such methods include preparing probes orprimers from the disclosed sequence and identifying or amplifying thecorresponding gene from appropriate sources of genomic material.

[0179] Also provided in the present invention are species homologs,allelic variants, and/or orthologs. The skilled artisan could, usingprocedures well-known in the art, obtain the polynucleotide sequencecorresponding to full-length genes (including, but not limited to thefull-length coding region), allelic variants, splice variants,orthologs, and/or species homologues of genes corresponding to SEQ IDNO:1, SEQ ID NO:2, or a deposited clone, relying on the sequence fromthe sequences disclosed herein or the clones deposited with the ATCC.For example, allelic variants and/or species homologues may be isolatedand identified by making suitable probes or primers which correspond tothe 5′, 3′, or internal regions of the sequences provided herein andscreening a suitable nucleic acid source for allelic variants and/or thedesired homologue.

[0180] The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0181] The polypeptides may be in the form of the protein, or may be apart of a larger protein, such as a fusion protein (see below). It isoften advantageous to include an additional amino acid sequence whichcontains secretory or leader sequences, pro-sequences, sequences whichaid in purification, such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

[0182] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

[0183] The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:1,and/or a cDNA provided in ATCC Deposit No. Z:. The present inventionalso provides a polypeptide comprising, or alternatively consisting of,the sequence identified as SEQ ID NO:2, and/or a polypeptide encoded bythe cDNA provided in ATCC Deposit NO:Z. The present invention alsoprovides polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:2,and/or a polypeptide sequence encoded by the cDNA contained in ATCCDeposit No:Z.

[0184] Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No.: that is lessthan, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000basepairs, 20,000 basepairs, or 10,000 basepairs in length.

[0185] The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:1, the sequence contained in a deposit, and/orthe nucleic acid sequence encoding the sequence disclosed as SEQ IDNO:2.

[0186] The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystringent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table II below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R. TABLE II HyridizationStringency Polynucleotide Hybrid Length Temperature and Wash TemperatureCondition Hybrid± (bp)‡ Buffer† and Buffer† A DNA:DNA > or equal to 5065° C.; 1xSSC - 65° C.; or-42° C.; 0.3xSSC 1xSSC, 50% formamide BDNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 50 67° C.;1xSSC - 67° C.; or-45° C.; 0.3xSSC 1xSSC, 50% formamide D DNA:RNA <50Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 50 70° C.; 1xSSC - 70° C.;or-50° C.; 0.3xSSC 1xSSC, 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*;1xSSC G DNA:DNA > or equal to 50 65° C.; 4xSSC - 65° C.; 1xSSC or-45°C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA >or equal to 50 67° C.; 4xSSC - 67° C.; 1xSSC or-45° C.; 4xSSC, 50%formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 5070° C.; 4xSSC - 67° C.; 1xSSC or-40° C.; 6xSSC, 50% formamide L RNA:RNA<50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50 50° C.; 4xSSC - 50°C.; 2xSSC or-40° C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*;6xSSC O DNA:RNA > or equal to 50 55° C.; 4xSSC - 55° C.; 2xSSC or-42°C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA >or equal to 50 60° C.; 4xSSC - 60° C.; 2xSSC or-45° C.; 6xSSC, 50%formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC # Methods of aligning twoor more polynucleotide sequences and/or determining the percent identitybetween two polynucleotide sequences are well known in the art (e.g.,MegAlign program of the DNA*Star suite of programs, etc). # For hybridsbetween 18 and 49 base pairs in length, Tm(° C.) = 81.5 ±16.6(log₁₀[Na+]) + 0.41 (% G + C) − (600/N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([NA+] for 1xSSC = .165M).

[0187] Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

[0188] Preferably, such hybridizing polynucleotides have at least 70%sequence identity (more preferably, at least 80% identity; and mostpreferably at least 90% or 95% identity) with the polynucleotide of thepresent invention to which they hybridize, where sequence identity isdetermined by comparing the sequences of the hybridizing polynucleotideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. The determination of identity is well known in the art,and discussed more specifically elsewhere herein.

[0189] The invention encompasses the application of PCR methodology tothe polynucleotide sequences of the present invention, the clonedeposited with the ATCC, and/or the cDNA encoding the polypeptides ofthe present invention. PCR techniques for the amplification of nucleicacids are described in U.S. Pat. No. 4,683,195 and Saiki et al.,Science, 239:487-491 (1988). PCR, for example, may include the followingsteps, of denaturation of template nucleic acid (if double-stranded),annealing of primer to target, and polymerization. The nucleic acidprobed or used as a template in the amplification reaction may begenomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specificsequences from genomic DNA, specific RNA sequence, and/or cDNAtranscribed from mRNA. References for the general use of PCR techniques,including specific method parameters, include Mullis et al., Cold SpringHarbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology,Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650,(1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds.,Innis et al., Academic Press, New York, (1990).

[0190] Signal Sequences

[0191] The present invention also encompasses mature forms of thepolypeptide comprising, or alternatively consisting of, the polypeptidesequence of SEQ ID NO:2, the polypeptide encoded by the polynucleotidedescribed as SEQ ID NO:1, and/or the polypeptide sequence encoded by acDNA in the deposited clone. The present invention also encompassespolynucleotides encoding mature forms of the present invention, such as,for example the polynucleotide sequence of SEQ ID NO:1, and/or thepolynucleotide sequence provided in a cDNA of the deposited clone.

[0192] According to the signal hypothesis, proteins secreted byeukaryotic cells have a signal or secretary leader sequence which iscleaved from the mature protein once export of the growing protein chainacross the rough endoplasmic reticulum has been initiated. Mosteukaryotic cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that cleavage specificity of a secretedprotein is ultimately determined by the primary structure of thecomplete protein, that is, it is inherent in the amino acid sequence ofthe polypeptide.

[0193] Methods for predicting whether a protein has a signal sequence,as well as the cleavage point for that sequence, are available. Forinstance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses theinformation from a short N-terminal charged region and a subsequentuncharged region of the complete (uncleaved) protein. The method of vonHeinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information fromthe residues surrounding the cleavage site, typically residues −13 to+2, where +1 indicates the amino terminus of the secreted protein. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. (vonHeinje, supra.) However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0194] The established method for identifying the location of signalsequences, in addition, to their cleavage sites has been the SignalPprogram (v1.1) developed by Henrik Nielsen et al., Protein Engineering10:1-6 (1997). The program relies upon the algorithm developed by vonHeinje, though provides additional parameters to increase the predictionaccuracy.

[0195] More recently, a hidden Markov model has been developed (H.Neilson, et al., Ismb 1998;6:122-30), which has been incorporated intothe more recent SignalP (v2.0). This new method increases the ability toidentify the cleavage site by discriminating between signal peptides anduncleaved signal anchors. The present invention encompasses theapplication of the method disclosed therein to the prediction of thesignal peptide location, including the cleavage site, to any of thepolypeptide sequences of the present invention.

[0196] As one of ordinary skill would appreciate, however, cleavagesites sometimes vary from organism to organism and cannot be predictedwith absolute certainty. Accordingly, the polypeptide of the presentinvention may contain a signal sequence. Polypeptides of the inventionwhich comprise a signal sequence have an N-terminus beginning within 5residues (i.e., + or −5 residues, or preferably at the −5, −4, −3, −2,−1, +1, +2, +3, +4, or +5 residue) of the predicted cleavage point.Similarly, it is also recognized that in some cases, cleavage of thesignal sequence from a secreted protein is not entirely uniform,resulting in more than one secreted species. These polypeptides, and thepolynucleotides encoding such polypeptides, are contemplated by thepresent invention.

[0197] Moreover, the signal sequence identified by the above analysismay not necessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:1 and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as describedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

[0198] Polynucleotide and Polypeptide Variants

[0199] The present invention also encompasses variants (e.g., allelicvariants, orthologs, etc.) of the polynucleotide sequence disclosedherein in SEQ ID NO:1, the complementary strand thereto, and/or the cDNAsequence contained in the deposited clone.

[0200] The present invention also encompasses variants of thepolypeptide sequence, and/or fragments therein, disclosed in SEQ IDNO:2, a polypeptide encoded by the polynucleotide sequence in SEQ IDNO:1, and/or a polypeptide encoded by a cDNA in the deposited clone.

[0201] “Variant” refers to a polynucleotide or polypeptide differingfrom the polynucleotide or polypeptide of the present invention, butretaining essential properties thereof. Generally, variants are overallclosely similar, and, in many regions, identical to the polynucleotideor polypeptide of the present invention.

[0202] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising, or alternatively consisting of, apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a HLRRSI1 relatedpolypeptide having an amino acid sequence as shown in the sequencelisting and described in SEQ ID NO:1 or the cDNA contained in ATCCdeposit No:Z; (b) a nucleotide sequence encoding a mature HLRRSI1related polypeptide having the amino acid sequence as shown in thesequence listing and described in SEQ ID NO:1 or the cDNA contained inATCC deposit No:Z; (c) a nucleotide sequence encoding a biologicallyactive fragment of a HLRRSI1 related polypeptide having an amino acidsequence shown in the sequence listing and described in SEQ ID NO:1 orthe cDNA contained in ATCC deposit No:Z; (d) a nucleotide sequenceencoding an antigenic fragment of a HLRRSI1 related polypeptide havingan amino acid sequence sown in the sequence listing and described in SEQID NO:1 or the cDNA contained in ATCC deposit No:Z; (e) a nucleotidesequence encoding a HLRRSI1 related polypeptide comprising the completeamino acid sequence encoded by a human cDNA plasmid contained in SEQ IDNO:1 or the cDNA contained in ATCC deposit No:Z; (f) a nucleotidesequence encoding a mature HLRRSI1 related polypeptide having an aminoacid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1or the cDNA contained in ATCC deposit No:Z; (g) a nucleotide sequenceencoding a biologically active fragment of a HLRRSI1 related polypeptidehaving an amino acid sequence encoded by a human cDNA plasmid containedin SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (h) anucleotide sequence encoding an antigenic fragment of a HLRRSI1 relatedpolypeptide having an amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC depositNo:Z; (I) a nucleotide sequence complimentary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

[0203] The present invention is also directed to polynucleotidesequences which comprise, or alternatively consist of, a polynucleotidesequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to, for example, any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.Polynucleotides encoded by these nucleic acid molecules are alsoencompassed by the invention. In another embodiment, the inventionencompasses nucleic acid molecules which comprise, or alternatively,consist of a polynucleotide which hybridizes under stringent conditions,or alternatively, under lower stringency conditions, to a polynucleotidein (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotideswhich hybridize to the complement of these nucleic acid molecules understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as arepolypeptides encoded by these polypeptides.

[0204] Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a HLRRSI1 related polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (b) a nucleotide sequence encoding a mature HLRRSI1 relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and descried in Table I; (c) a nucleotide sequence encoding abiologically active fragment of a HLRRSI1 related polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (d) a nucleotide sequence encoding an antigenic fragment of aHLRRSI1 related polypeptide having an amino acid sequence as shown inthe sequence listing and descried in Table I; (e) a nucleotide sequenceencoding a HLRRSI1 related polypeptide comprising the complete aminoacid sequence encoded by a human cDNA in a cDNA plasmid contained in theATCC Deposit and described in Table I; (f) a nucleotide sequenceencoding a mature HLRRSI1 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table I: (g) a nucleotide sequence encoding abiologically active fragment of a HLRRSI1 related polypeptide having anamino acid sequence encoded by a human cDNA in a cDNA plasmid containedin the ATCC Deposit and described in Table I; (h) a nucleotide sequenceencoding an antigenic fragment of a HLRRSI1 related polypeptide havingan amino acid sequence encoded by a human cDNA in a cDNA plasmidcontained in the ATCC deposit and described in Table I; (i) a nucleotidesequence complimentary to any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), (g), or (h) above.

[0205] The present invention is also directed to nucleic acid moleculeswhich comprise, or alternatively, consist of, a nucleotide sequencewhich is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to, for example, any of the nucleotide sequences in(a), (b), (c), (d), (e), (f), (g), or (h), above.

[0206] The present invention encompasses polypeptide sequences whichcomprise, or alternatively consist of, an amino acid sequence which isat least 80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to, the following non-limited examples, the polypeptidesequence identified as SEQ ID NO:2, the polypeptide sequence encoded bya cDNA provided in the deposited clone, and/or polypeptide fragments ofany of the polypeptides provided herein. Polynucleotides encoded bythese nucleic acid molecules are also encompassed by the invention. Inanother embodiment, the invention encompasses nucleic acid moleculeswhich comprise, or alternatively, consist of a polynucleotide whichhybridizes under stringent conditions, or alternatively, under lowerstringency conditions, to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), or (h), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolypeptides.

[0207] The present invention is also directed to polypeptides whichcomprise, or alternatively consist of, an amino acid sequence which isat least 80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to, for example, the polypeptide sequence shown in SEQ IDNO:2, a polypeptide sequence encoded by the nucleotide sequence in SEQID NO:1, a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z,and/or polypeptide fragments of any of these polypeptides (e.g., thosefragments described herein). Polynucleotides which hybridize to thecomplement of the nucleic acid molecules encoding these polypeptidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompasses by the present invention, asare the polypeptides encoded by these polynucleotides.

[0208] By a nucleic acid having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thenucleic acid is identical to the reference sequence except that thenucleotide sequence may include up to five point mutations per each 100nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence referenced in Table I, the ORF (open reading frame),or any fragment specified as described herein.

[0209] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

[0210] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

[0211] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the CLUSTALW alignmentdoes not show a matched/alignment of the first 10 bases at 5′ end. The10 unpaired bases represent 10% of the sequence (number of bases at the5′ and 3′ ends not matched/total number of bases in the query sequence)so 10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

[0212] In addition to the above method of aligning two or morepolynucleotide or polypeptide sequences to arrive at a percent identityvalue for the aligned sequences, it may be desirable in somecircumstances to use a modified version of the CLUSTALW algorithm whichtakes into account known structural features of the sequences to bealigned, such as for example, the SWISS-PROT designations for eachsequence. The result of such a modifed CLUSTALW algorithm may provide amore accurate value of the percent identity for two polynucleotide orpolypeptide sequences. Support for such a modified version of CLUSTALWis provided within the CLUSTALW algorithm and would be readilyappreciated to one of skill in the art of bioinformatics.

[0213] The variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in themRNA to those preferred by a bacterial host such as E. coli).

[0214] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level and are included inthe present invention. Alternatively, non-naturally occurring variantsmay be produced by mutagenesis techniques or by direct synthesis.

[0215] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem . . . 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

[0216] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem.268:22105-22111 (1993)) conducted extensive mutational analysis of humancytokine IL-1a. They used random mutagenesis to generate over 3,500individual IL-1a mutants that averaged 2.5 amino acid changes pervariant over the entire length of the molecule. Multiple mutations wereexamined at every possible amino acid position. The investigators foundthat “[m]ost of the molecule could be altered with little effect oneither [binding or biological activity].” In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0217] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

[0218] Alternatively, such N-terminus or C-terminus deletions of apolypeptide of the present invention may, in fact, result in asignificant increase in one or more of the biological activities of thepolypeptide(s). For example, biological activity of many polypeptidesare governed by the presence of regulatory domains at either one or bothtermini. Such regulatory domains effectively inhibit the biologicalactivity of such polypeptides in lieu of an activation event (e.g.,binding to a cognate ligand or receptor, phosphorylation, proteolyticprocessing, etc.). Thus, by eliminating the regulatory domain of apolypeptide, the polypeptide may effectively be rendered biologicallyactive in the absence of an activation event.

[0219] Thus, the invention further includes polypeptide variants thatshow substantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

[0220] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0221] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0222] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved.

[0223] The invention encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the polypeptide of the presentinvention. Similarity is determined by conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a polypeptide by another amino acid of like characteristics(e.g., chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0224] Tolerated conservative amino acid substitutions of the presentinvention involve replacement of the aliphatic or hydrophobic aminoacids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Serand Thr; replacement of the acidic residues Asp and Glu; replacement ofthe amide residues Asn and Gln, replacement of the basic residues Lys,Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp,and replacement of the small-sized amino acids Ala, Ser, Thr, Met, andGly.

[0225] In addition, the present invention also encompasses theconservative substitutions provided in Table III below. TABLE III ForAmino Acid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala,L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met,Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu,D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln,D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine QD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, AcpIsoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D- Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D-or L-1- oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D- Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D- Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

[0226] Aside from the uses described above, such amino acidsubstitutions may also increase protein or peptide stability. Theinvention encompasses amino acid substitutions that contain, forexample, one or more non-peptide bonds (which replace the peptide bonds)in the protein or peptide sequence. Also included are substitutions thatinclude amino acid residues other than naturally occurring L-aminoacids, e.g., D-amino acids or non-naturally occurring or synthetic aminoacids, e.g., B or y amino acids.

[0227] Both identity and similarity can be readily calculated byreference to the following publications: Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Informatics Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0228] In addition, the present invention also encompasses substitutionof amino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

[0229] Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

[0230] Besides conservative amino acid substitution, variants of thepresent invention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0231] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0232] Moreover, the invention further includes polypeptide variantscreated through the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:1, the sequenceof the clone submitted in a deposit, and/or the cDNA encoding thepolypeptide disclosed as SEQ ID NO:2. Such DNA Shuffling technology isknown in the art and more particularly described elsewhere herein (e.g.,WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples providedherein).

[0233] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of the present invention havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of the present invention, which contains atleast one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of thepresent invention or fragments thereof (e.g., the mature form and/orother fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or50-150, conservative amino acid substitutions are preferable.

[0234] Polynucleotide and Polypeptide Fragments

[0235] The present invention is directed to polynucleotide fragments ofthe polynucleotides of the invention, in addition to polypeptidesencoded therein by said polynucleotides and/or fragments.

[0236] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence which: is a portionof that contained in a deposited clone, or encoding the polypeptideencoded by the cDNA in a deposited clone; is a portion of that shown inSEQ ID NO:1 or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:2. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases from the cDNA sequence containedin a deposited clone or the nucleotide sequence shown in SEQ ID NO:1. Inthis context “about” includes the particularly recited value, a valuelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus, or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

[0237] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ IDNO:1, or the complementary strand thereto, or the cDNA contained in adeposited clone. In this context “about” includes the particularlyrecited ranges, and ranges larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity. Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein. Also encompassed by the present invention arepolynucleotides which hybridize to these nucleic acid molecules understringent hybridization conditions or lower stringency conditions, asare the polypeptides encoded by these polynucleotides.

[0238] In the present invention, a “polypeptide fragment” refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:2or encoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0239] Preferred polypeptide fragments include the full-length protein.Further preferred polypeptide fragments include the full-length proteinhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. For example, any number of amino acids,ranging from 1-60, can be deleted from the amino terminus of thefull-length polypeptide. Similarly, any number of amino acids, rangingfrom 1-30, can be deleted from the carboxy terminus of the full-lengthprotein. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

[0240] Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2 falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

[0241] Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity. Polynucleotides encoding these polypeptidefragments are also encompassed by the invention.

[0242] In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

[0243] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. Z or encoded by a polynucleotide that hybridizes to the complementof the sequence of SEQ ID NO:1 or contained in ATCC deposit No. Z understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNO:1), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

[0244] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0245] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0246] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0247] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0248] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0249] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

[0250] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:1 and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0251] Antibodies

[0252] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, monovalent, bispecific, heteroconjugate, multispecific,human, humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. The term “antibody,” as used herein,refers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule. Moreover, the term“antibody” (Ab) or “monoclonal antibody” (Mab) is meant to includeintact molecules, as well as, antibody fragments (such as, for example,Fab and F(ab′)₂ fragments) which are capable of specifically binding toprotein. Fab and F(ab′)₂ fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation of the animal orplant, and may have less non-specific tissue binding than an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred, as well as the products of a FAB or otherimmunoglobulin expression library. Moreover, antibodies of the presentinvention include chimeric, single chain, and humanized antibodies.

[0253] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0254] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0255] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0256] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homologue of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologuesof human proteins and the corresponding epitopes thereof. Antibodiesthat do not bind polypeptides with less than 95%, less than 90%, lessthan 85%, less than 80%, less than 75%, less than 70%, less than 65%,less than 60%, less than 55%, and less than 50% identity (as calculatedusing methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further included in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M,5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15M, or 10-15 M.

[0257] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0258] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0259] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem . . . 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0260] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0261] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

[0262] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0263] The antibodies of the present invention may be generated by anysuitable method known in the art.

[0264] The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988), which is herebyincorporated herein by reference in its entirety). For example, apolypeptide of the invention can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc. to induce theproduction of sera containing polyclonal antibodies specific for theantigen. The administration of the polypeptides of the present inventionmay entail one or more injections of an immunizing agent and, ifdesired, an adjuvant. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art. For the purposesof the invention, “immunizing agent” may be defined as a polypeptide ofthe invention, including fragments, variants, and/or derivativesthereof, in addition to fusions with heterologous polypeptides and otherforms of the polypeptides described herein.

[0265] Typically, the immunizing agent and/or adjuvant will be injectedin the mammal by multiple subcutaneous or intraperitoneal injections,though they may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0266] The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., (1981)), or other methods known to theartisan. Other examples of methods which may be employed for producingmonoclonal antibodies includes, but are not limited to, the human B-cellhybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole etal., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and theEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies AndCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the mAb of this invention maybe cultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

[0267] In a hybridoma method, a mouse, a humanized mouse, a mouse with ahuman immune system, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

[0268] The immunizing agent will typically include polypeptides of thepresent invention or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes (“PBLs”) are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986), pp.59-103). Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

[0269] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. As inferred throughout the specification,human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York,(1987) pp. 51-63).

[0270] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst the polypeptides of the present invention. Preferably, thebinding specificity of monoclonal antibodies produced by the hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbantassay (ELISA). Such techniques are known in the art and within the skillof the artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

[0271] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods (Goding, supra). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

[0272] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-sepharose, hydroxyapatite chromatography, gelexclusion chromatography, gel electrophoresis, dialysis, or affinitychromatography.

[0273] The skilled artisan would acknowledge that a variety of methodsexist in the art for the production of monoclonal antibodies and thus,the invention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

[0274] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0275] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art. Monoclonal antibodies can be prepared usinga wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

[0276] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples herein. In a non-limiting example,mice can be immunized with a polypeptide of the invention or a cellexpressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well-known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

[0277] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0278] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)₂ fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0279] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0280] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

[0281] For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

[0282] In general, the humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992).

[0283] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety. The techniques of coleet al., and Boerder et al., are also available for the preparation ofhuman monoclonal antibodies (cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

[0284] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

[0285] Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

[0286] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0287] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0288] The antibodies of the present invention may be bispecificantibodies. Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present invention, one of the bindingspecificities may be directed towards a polypeptide of the presentinvention, the other may be for any other antigen, and preferably for acell-surface protein, receptor, receptor subunit, tissue-specificantigen, virally derived protein, virally encoded envelope protein,bacterially derived protein, or bacterial surface protein, etc.

[0289] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0290] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

[0291] Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0292] Polynucleotides Encoding Antibodies

[0293] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2.

[0294] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0295] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0296] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0297] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0298] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0299] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

[0300] Methods of Producing Antibodies

[0301] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0302] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0303] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0304] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0305] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem . .. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0306] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0307] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0308] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0309] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0310] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0311] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0312] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0313] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0314] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439, 095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0315] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0316] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2 may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem . . . 270:9459-9471 (1995).

[0317] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0318] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

[0319] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

[0320] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0321] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0322] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0323] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0324] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic. The present invention alsoencompasses the creation of synthetic antibodies directed against thepolypeptides of the present invention. One example of syntheticantibodies is described in Radrizzani, M., et al., Medicina, (Aires),59(6):753-8, (1999)). Recently, a new class of synthetic antibodies hasbeen described and are referred to as molecularly imprinted polymers(MIPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often usedas molecular recognition elements in chemical and biological sensors.However, their lack of stability and signal transduction mechanismslimits their use as sensing devices. Molecularly imprinted polymers(MIPs) are capable of mimicking the function of biological receptors butwith less stability constraints. Such polymers provide high sensitivityand selectivity while maintaining excellent thermal and mechanicalstability. MIPs have the ability to bind to small molecules and totarget molecules such as organics and proteins' with equal or greaterpotency than that of natural antibodies. These “super” MIPs have higheraffinities for their target and thus require lower concentrations forefficacious binding. During synthesis, the MIPs are imprinted so as tohave complementary size, shape, charge and functional groups of theselected target by using the target molecule itself (such as apolypeptide, antibody, etc.), or a substance having a very similarstructure, as its “print” or “template.” MIPs can be derivatized withthe same reagents afforded to antibodies. For example, fluorescent‘super’ MIPs can be coated onto beads or wells for use in highlysensitive separations or assays, or for use in high throughput screeningof proteins.

[0325] Moreover, MIPs based upon the structure of the polypeptide(s) ofthe present invention may be useful in screening for compounds that bindto the polypeptide(s) of the invention. Such a MIP would serve the roleof a synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

[0326] MIPs have also been shown to be useful in “sensing” the presenceof its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens,Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J.L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J.L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed usinga polypeptide of the present invention may be used in assays designed toidentify, and potentially quantitate, the level of said polypeptide in asample. Such a MIP may be used as a substitute for any componentdescribed in the assays, or kits, provided herein (e.g., ELISA, etc.).

[0327] A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

[0328] Uses for Antibodies Directed Against Polypeptides of theInvention

[0329] The antibodies of the present invention have various utilities.For example, such antibodies may be used in diagnostic assays to detectthe presence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

[0330] Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

[0331] Antibodies directed against the polypeptides of the presentinvention are useful for the affinity purification of such polypeptidesfrom recombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

[0332] Immunophenotyping

[0333] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0334] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0335] Assays For Antibody Binding

[0336] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0337] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0338] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0339] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0340] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

[0341] Therapeutic Uses of Antibodies

[0342] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0343] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0344] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0345] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0346] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

[0347] Antibodies directed against polypeptides of the present inventionare useful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

[0348] Likewise, one could envision cloning the gene encoding anantibody directed against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

[0349] Alternatively, antibodies of the present invention could beproduced in a plant (e.g., cloning the gene of the antibody directedagainst a polypeptide of the present invention, and transforming a plantwith a suitable vector comprising said gene for constitutive expressionof the antibody within the plant), and the plant subsequently ingestedby an animal, thereby conferring temporary immunity to the animal forthe specific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

[0350] In another embodiment, antibodies of the present invention,preferably polyclonal antibodies, more preferably monoclonal antibodies,and most preferably single-chain antibodies, can be used as a means ofinhibiting gene expression of a particular gene, or genes, in a human,mammal, and/or other organism. See, for example, InternationalPublication Number WO 00/05391, published Feb. 3, 2000, to DowAgrosciences LLC. The application of such methods for the antibodies ofthe present invention are known in the art, and are more particularlydescribed elsewhere herein.

[0351] In yet another embodiment, antibodies of the present inventionmay be useful for multimerizing the polypeptides of the presentinvention. For example, certain proteins may confer enhanced biologicalactivity when present in a multimeric state (i.e., such enhancedactivity may be due to the increased effective concentration of suchproteins whereby more protein is available in a localized location).

[0352] Antibody-Based Gene Therapy

[0353] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0354] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0355] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0356] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0357] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0358] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem . . . 262:4429-4432(1987)) (which can be used to target cell types specifically expressingthe receptors), etc. In another embodiment, nucleic acid-ligandcomplexes can be formed in which the ligand comprises a fusogenic viralpeptide to disrupt endosomes, allowing the nucleic acid to avoidlysosomal degradation. In yet another embodiment, the nucleic acid canbe targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Publications WO 92/06180;WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, thenucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression, by homologous recombination (Koller andSmithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra etal., Nature 342:435-438 (1989)).

[0359] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0360] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0361] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0362] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0363] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0364] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0365] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0366] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0367] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0368] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0369] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0370] Therapeutic/Prophylactic Administration and Compositions

[0371] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0372] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0373] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0374] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0375] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0376] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0377] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0378] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0379] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0380] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0381] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0382] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0383] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0384] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0385] Diagnosis and Imaging with Antibodies

[0386] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

[0387] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0388] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0389] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0390] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0391] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0392] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0393] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0394] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0395] Kits

[0396] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0397] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0398] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0399] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0400] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0401] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0402] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0403] Fusion Proteins

[0404] Any polypeptide of the present invention can be used to generatefusion proteins. For example, the polypeptide of the present invention,when fused to a second protein, can be used as an antigenic tag.Antibodies raised against the polypeptide of the present invention canbe used to indirectly detect the second protein by binding to thepolypeptide. Moreover, because certain proteins target cellularlocations based on trafficking signals, the polypeptides of the presentinvention can be used as targeting molecules once fused to otherproteins.

[0405] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0406] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

[0407] Moreover, polypeptides of the present invention, includingfragments, and specifically epitopes, can be combined with parts of theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).) Similarly, EP-A-O 464 533(Canadian counterpart 2045869) discloses fusion proteins comprisingvarious portions of the constant region of immunoglobulin moleculestogether with another human protein or part thereof. In many cases, theFc part in a fusion protein is beneficial in therapy and diagnosis, andthus can result in, for example, improved pharmacokinetic properties.(EP-A 0232 262.) Alternatively, deleting the Fc part after the fusionprotein has been expressed, detected, and purified, would be desired.For example, the Fc portion may hinder therapy and diagnosis if thefusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, D. Bennett et al., J. MolecularRecognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem . . .270:9459-9471 (1995).)

[0408] Moreover, the polypeptides of the present invention can be fusedto marker sequences (also referred to as “tags”). Due to theavailability of antibodies specific to such “tags”, purification of thefused polypeptide of the invention, and/or its identification issignificantly facilitated since antibodies specific to the polypeptidesof the invention are not required. Such purification may be in the formof an affinity purification whereby an anti-tag antibody or another typeof affinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

[0409] The skilled artisan would acknowledge the existence of other“tags” which could be readily substituted for the tags referred to suprafor purification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ IDNO:27), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem . . . , 266:15136-15166, (1991));the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.Sci. USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

[0410] The present invention also encompasses the attachment of up tonine codons encoding a repeating series of up to nine arginine aminoacids to the coding region of a polynucleotide of the present invention.The invention also encompasses chemically derivitizing a polypeptide ofthe present invention with a repeating series of up to nine arginineamino acids. Such a tag, when attached to a polypeptide, has recentlybeen shown to serve as a universal pass, allowing compounds access tothe interior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

[0411] Protein fusions involving polypeptides of the present invention,including fragments and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

[0412] Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be determined, toxins, such as ricin,saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999;886:233-5), or HCtoxin (Tonukari N J, et al., Plant Cell. 2000 February;12(2):237-248),for example. Such fusions could be used to deliver the toxins to desiredtissues for which a ligand or a protein capable of binding to thepolypeptides of the invention exists.

[0413] The invention encompasses the fusion of antibodies directedagainst polypeptides of the present invention, including variants andfragments thereof, to said toxins for delivering the toxin to specificlocations in a cell, to specific tissues, and/or to specific species.Such bifunctional antibodies are known in the art, though a reviewdescribing additional advantageous fusions, including citations formethods of production, can be found in P. J. Hudson, Curr. Opp. In. 1mm. 11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fungal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

[0414] Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

[0415] Vectors, Host Cells, and Protein Production

[0416] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0417] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0418] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0419] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

[0420] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPA0815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

[0421] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

[0422] A polypeptide of this invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0423] Polypeptides of the present invention, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0424] In one embodiment, the yeast Pichia pastoris is used to expressthe polypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

[0425] In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

[0426] Many other yeast vectors could be used in place of pPIC9K, suchas, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

[0427] In another embodiment, high-level expression of a heterologouscoding sequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

[0428] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with the polynucleotides of the invention,and which activates, alters, and/or amplifies endogenouspolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous polynucleotide sequences via homologousrecombination, resulting in the formation of a new transcription unit(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.5,733,761, issued Mar. 31, 1998; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

[0429] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). Forexample, a polypeptide corresponding to a fragment of a polypeptidesequence of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the polypeptide sequence. Non-classical amino acidsinclude, but are not limited to, to the D-isomers of the common aminoacids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid,Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0430] The invention encompasses polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

[0431] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N- or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

[0432] Also provided by the invention are chemically modifiedderivatives of the polypeptides of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0433] The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

[0434] The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

[0435] For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0436] Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

[0437] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0438] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0439] As with the various polymers exemplified above, it iscontemplated that the polymeric residues may contain functional groupsin addition, for example, to those typically involved in linking thepolymeric residues to the polypeptides of the present invention. Suchfunctionalities include, for example, carboxyl, amine, hydroxy and thiolgroups. These functional groups on the polymeric residues can be furtherreacted, if desired, with materials that are generally reactive withsuch functional groups and which can assist in targeting specifictissues in the body including, for example, diseased tissue. Exemplarymaterials which can be reacted with the additional functional groupsinclude, for example, proteins, including antibodies, carbohydrates,peptides, glycopeptides, glycolipids, lectins, and nucleosides.

[0440] In addition to residues of hydrophilic polymers, the chemicalused to derivatize the polypeptides of the present invention can be asaccharide residue. Exemplary saccharides which can be derived include,for example, monosaccharides or sugar alcohols, such as erythrose,threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitoland sedoheptulose, with preferred monosaccharides being fructose,mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides,such as lactose, sucrose, maltose and cellobiose. Other saccharidesinclude, for example, inositol and ganglioside head groups. Othersuitable saccharides, in addition to those exemplified above, will bereadily apparent to one skilled in the art based on the presentdisclosure. Generally, saccharides which may be used for derivitizationinclude saccharides that can be attached to the polypeptides of theinvention via alkylation or acylation reactions.

[0441] Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

[0442] The invention encompasses derivitization of the polypeptides ofthe present invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof. Accordingly, suitable polymers include, forexample, proteins, such as albumin, polyalginates, andpolylactide-coglycolide polymers. Exemplary semi-synthetic polymersinclude carboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as Pluronics.RTM., commercially available from BASF,Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof. Methods for thepreparation of derivatized polypeptides of the invention which employpolymers as stabilizing compounds will be readily apparent to oneskilled in the art, in view of the present disclosure, when coupled withinformation known in the art, such as that described and referred to inUnger, U.S. Pat. No. 5,205,290, the disclosure of which is herebyincorporated by reference herein in its entirety.

[0443] Moreover, the invention encompasses additional modifications ofthe polypeptides of the present invention. Such additional modificationsare known in the art, and are specifically provided, in addition tomethods of derivitization, etc., in U.S. Pat. No. 6,028,066, which ishereby incorporated in its entirety herein.

[0444] The polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of thepolypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0445] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only polypeptides corresponding to the amino acid sequence ofSEQ ID NO:2 or encoded by the cDNA contained in a deposited clone(including fragments, variants, splice variants, and fusion proteins,corresponding to these polypeptides as described herein). These homomersmay contain polypeptides having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

[0446] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the polypeptides of the invention. Ina specific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

[0447] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

[0448] In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained inan Fc fusion protein of the invention (as described herein). In anotherspecific example, covalent associations of fusion proteins of theinvention are between heterologous polypeptide sequence from anotherprotein that is capable of forming covalently associated multimers, suchas for example, osteoprotegerin (see, e.g., International PublicationNO: WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or morepolypeptides of the invention are joined through peptide linkers.Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple polypeptides of the invention separated by peptide linkers maybe produced using conventional recombinant DNA technology.

[0449] Another method for preparing multimer polypeptides of theinvention involves use of polypeptides of the invention fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

[0450] Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

[0451] In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

[0452] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0453] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhydrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0454] In addition, the polynucleotide insert of the present inventioncould be operatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

[0455] Uses of the Polynucleotides

[0456] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0457] The polynucleotides of the present invention are useful forchromosome identification. There exists an ongoing need to identify newchromosome markers, since few chromosome marking reagents, based onactual sequence data (repeat polymorphisms), are presently available.Each polynucleotide of the present invention can be used as a chromosomemarker.

[0458] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1.Primers can be selected using computer analysis so that primers do notspan more than one predicted exon in the genomic DNA. These primers arethen used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the SEQ ID NO:1 will yield an amplified fragment.

[0459] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

[0460] Precise chromosomal location of the polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

[0461] For chromosome mapping, the polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0462] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

[0463] Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

[0464] Furthermore, increased or decreased expression of the gene inaffected organisms as compared to unaffected organisms can be assessedusing polynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

[0465] Thus, the invention also provides a diagnostic method usefulduring diagnosis of a disorder, involving measuring the expression levelof polynucleotides of the present invention in cells or body fluid froman organism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

[0466] By “measuring the expression level of a polynucleotide of thepresent invention” is intended qualitatively or quantitatively measuringor estimating the level of the polypeptide of the present invention orthe level of the mRNA encoding the polypeptide in a first biologicalsample either directly (e.g., by determining or estimating absoluteprotein level or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

[0467] By “biological sample” is intended any biological sample obtainedfrom an organism, body fluids, cell line, tissue culture, or othersource which contains the polypeptide of the present invention or mRNA.As indicated, biological samples include body fluids (such as thefollowing non-limiting examples, sputum, amniotic fluid, urine, saliva,breast milk, secretions, interstitial fluid, blood, serum, spinal fluid,etc.) which contain the polypeptide of the present invention, and othertissue sources found to express the polypeptide of the presentinvention. Methods for obtaining tissue biopsies and body fluids fromorganisms are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

[0468] The method(s) provided above may Preferably be applied in adiagnostic method and/or kits in which polynucleotides and/orpolypeptides are attached to a solid support. In one exemplary method,the support may be a “gene chip” or a “biological chip” as described inU.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a genechip with polynucleotides of the present invention attached may be usedto identify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supraare hereby incorporated by reference in their entirety herein.

[0469] The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

[0470] In addition to the foregoing, a polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Antisense techniques are discussed, for example, in Okano, J.Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed in, for instance Lee et al., NucleicAcids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);and Dervan et al., Science 251: 1360 (1991). Both methods rely onbinding of the polynucleotide to a complementary DNA or RNA. For thesetechniques, preferred polynucleotides are usually oligonucleotides 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

[0471] The present invention encompasses the addition of a nuclearlocalization signal, operably linked to the 5′ end, 3′ end, or anylocation therein, to any of the oligonucleotides, antisenseoligonucleotides, triple helix oligonucleotides, ribozymes, PNAoligonucleotides, and/or polynucleotides, of the present invention. See,for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000);which is hereby incorporated herein by reference.

[0472] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

[0473] The polynucleotides are also useful for identifying organismsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The polynucleotides of the presentinvention can be used as additional DNA markers for RFLP.

[0474] The polynucleotides of the present invention can also be used asan alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an organisms genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,organisms can be identified because each organism will have a unique setof DNA sequences. Once an unique ID database is established for anorganism, positive identification of that organism, living or dead, canbe made from extremely small tissue samples. Similarly, polynucleotidesof the present invention can be used as polymorphic markers, in additionto, the identification of transformed or non-transformed cells and/ortissues.

[0475] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from the sequences of the present invention. Panels ofsuch reagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination. Moreover, as mentioned above, such reagents can beused to screen and/or identify transformed and non-transformed cellsand/or tissues.

[0476] In the very least, the polynucleotides of the present inventioncan be used as molecular weight markers on Southern gels, as diagnosticprobes for the presence of a specific mRNA in a particular cell type, asa probe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

[0477] Uses of the Polypeptides

[0478] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0479] A polypeptide of the present invention can be used to assayprotein levels in a biological sample using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0480] In addition to assaying protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0481] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, 112In, 99mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99 mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).) Thus, the invention provides a diagnosticmethod of a disorder, which involves (a) assaying the expression of apolypeptide of the present invention in cells or body fluid of anindividual; (b) comparing the level of gene expression with a standardgene expression level, whereby an increase or decrease in the assayedpolypeptide gene expression level compared to the standard expressionlevel is indicative of a disorder. With respect to cancer, the presenceof a relatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0482] Moreover, polypeptides of the present invention can be used totreat, prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

[0483] Similarly, antibodies directed to a polypeptide of the presentinvention can also be used to treat, prevent, and/or diagnose disease.For example, administration of an antibody directed to a polypeptide ofthe present invention can bind and reduce overproduction of thepolypeptide. Similarly, administration of an antibody can activate thepolypeptide, such as by binding to a polypeptide bound to a membrane(receptor).

[0484] At the very least, the polypeptides of the present invention canbe used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. Polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, thepolypeptides of the present invention can be used to test the followingbiological activities.

[0485] Gene Therapy Methods

[0486] Another aspect of the present invention is to gene therapymethods for treating or preventing disorders, diseases and conditions.The gene therapy methods relate to the introduction of nucleic acid(DNA, RNA and antisense DNA or RNA) sequences into an animal to achieveexpression of a polypeptide of the present invention. This methodrequires a polynucleotide which codes for a polypeptide of the inventionthat operatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

[0487] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

[0488] As discussed in more detail below, the polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). Thepolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0489] In one embodiment, the polynucleotide of the invention isdelivered as a naked polynucleotide. The term “naked” polynucleotide,DNA or RNA refers to sequences that are free from any delivery vehiclethat acts to assist, promote or facilitate entry into the cell,including viral sequences, viral particles, liposome formulations,lipofectin or precipitating agents and the like. However, thepolynucleotides of the invention can also be delivered in liposomeformulations and lipofectin formulations and the like can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

[0490] The polynucleotide vector constructs of the invention used in thegene therapy method are preferably constructs that will not integrateinto the host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0491] Any strong promoter known to those skilled in the art can be usedfor driving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

[0492] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0493] The polynucleotide construct of the invention can be delivered tothe interstitial space of tissues within the an animal, including ofmuscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart,lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0494] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[0495] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0496] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0497] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0498] In certain embodiments, the polynucleotide constructs of theinvention are complexed in a liposome preparation. Liposomalpreparations for use in the instant invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7416 (1987), which is hereinincorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.USA, 86:6077-6081 (1989), which is herein incorporated by reference);and purified transcription factors (Debs et al., J. Biol. Chem . . . ,265:10189-10192 (1990), which is herein incorporated by reference), infunctional form.

[0499] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0500] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0501] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0502] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0503] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem . . . , 255:10431 (1980); Szoka et al., Proc. Natl.Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

[0504] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0505] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication NO: WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0506] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding polypeptides of the invention. Retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus,and mammary tumor virus.

[0507] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14×, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO4 precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0508] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding polypeptides of theinvention. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express polypeptides of the invention.

[0509] In certain other embodiments, cells are engineered, ex vivo or invivo, with polynucleotides of the invention contained in an adenovirusvector. Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

[0510] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0511] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[0512] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0513] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

[0514] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

[0515] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the desired endogenous polynucleotidesequence so the promoter will be operably linked to the endogenoussequence upon homologous recombination.

[0516] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0517] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0518] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

[0519] The polynucleotides encoding polypeptides of the presentinvention may be administered along with other polynucleotides encodingangiogenic proteins. Angiogenic proteins include, but are not limitedto, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C),VEGF-3 (VEGF-B), epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

[0520] Preferably, the polynucleotide encoding a polypeptide of theinvention contains a secretory signal sequence that facilitatessecretion of the protein. Typically, the signal sequence is positionedin the coding region of the polynucleotide to be expressed towards or atthe 5′ end of the coding region. The signal sequence may be homologousor heterologous to the polynucleotide of interest and may be homologousor heterologous to the cells to be transfected. Additionally, the signalsequence may be chemically synthesized using methods known in the art.

[0521] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers. (Kaneda et al., Science, 243:375 (1989)).

[0522] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0523] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0524] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0525] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281(1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0526] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

[0527] Biological Activities

[0528] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

[0529] Immune Activity

[0530] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

[0531] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

[0532] Moreover, a polynucleotides or polypeptides, or agonists orantagonists of the present invention could also be used to modulatehemostatic (the stopping of bleeding) or thrombolytic activity (clotformation). For example, by increasing hemostatic or thrombolyticactivity, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could be used to treat or prevent bloodcoagulation diseases, disorders, and/or conditions (e.g.,afibrinogenemia, factor deficiencies, arterial thrombosis, venousthrombosis, etc.), blood platelet diseases, disorders, and/or conditions(e.g. thrombocytopenia), or wounds resulting from trauma, surgery, orother causes. Alternatively, a polynucleotides or polypeptides, oragonists or antagonists of the present invention that can decreasehemostatic or thrombolytic activity could be used to inhibit or dissolveclotting. Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention are may also be useful for the detection,prognosis, treatment, and/or prevention of heart attacks (infarction),strokes, scarring, fibrinolysis, uncontrolled bleeding, uncontrolledcoagulation, uncontrolled complement fixation, and/or inflammation.

[0533] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

[0534] Examples of autoimmune diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed or detected by thepresent invention include, but are not limited to: Addison's Disease,hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

[0535] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

[0536] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be used to treat, prevent, and/ordiagnose organ rejection or graft-versus-host disease (GVHD). Organrejection occurs by host immune cell destruction of the transplantedtissue through an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

[0537] Similarly, a polynucleotides or polypeptides, or agonists orantagonists of the present invention may also be used to modulateinflammation. For example, the polypeptide or polynucleotide or agonistsor antagonist may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat, prevent, and/or diagnose inflammatory conditions, both chronicand acute conditions, including chronic prostatitis, granulomatousprostatitis and malacoplakia, inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

[0538] Hyperproliferative Disorders

[0539] A polynucleotides or polypeptides, or agonists or antagonists ofthe invention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

[0540] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

[0541] Examples of hyperproliferative diseases, disorders, and/orconditions that can be treated, prevented, and/or diagnosed bypolynucleotides or polypeptides, or agonists or antagonists of thepresent invention include, but are not limited to neoplasms located inthe: colon, abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

[0542] Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

[0543] One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

[0544] Thus, the present invention provides a method for treating orpreventing cell proliferative diseases, disorders, and/or conditions byinserting into an abnormally proliferating cell a polynucleotide of thepresent invention, wherein said polynucleotide represses saidexpression.

[0545] Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

[0546] Polynucleotides of the present invention may be useful inrepressing expression of oncogenic genes or antigens. By “repressingexpression of the oncogenic genes” is intended the suppression of thetranscription of the gene, the degradation of the gene transcript(pre-message RNA), the inhibition of splicing, the destruction of themessenger RNA, the prevention of the post-translational modifications ofthe protein, the destruction of the protein, or the inhibition of thenormal function of the protein.

[0547] For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

[0548] The polynucleotides of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. Thepolynucleotides of the present invention may also be administered todisease sites at the time of surgical intervention.

[0549] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[0550] Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

[0551] The present invention is further directed to antibody-basedtherapies which involve administering of anti-polypeptides andanti-polynucleotide antibodies to a mammalian, preferably human, patientfor treating, preventing, and/or diagnosing one or more of the describeddiseases, disorders, and/or conditions. Methods for producinganti-polypeptides and anti-polynucleotide antibodies polyclonal andmonoclonal antibodies are described in detail elsewhere herein. Suchantibodies may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein.

[0552] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0553] In particular, the antibodies, fragments and derivatives of thepresent invention are useful for treating, preventing, and/or diagnosinga subject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

[0554] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

[0555] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of diseases, disorders,and/or conditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-6M,10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M,5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M,5×10-15M, and 10-15M.

[0556] Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

[0557] Polypeptides, including protein fusions, of the presentinvention, or fragments thereof may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Saidpolypeptides may act either directly, or indirectly to induce apoptosisof proliferative cells and tissues, for example in the activation of adeath-domain receptor, such as tumor necrosis factor (TNF) receptor-1,CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein(TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and-2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998),which is hereby incorporated by reference). Moreover, in anotherpreferred embodiment of the present invention, said polypeptides mayinduce apoptosis through other mechanisms, such as in the activation ofother proteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses. 50(5):423-33 (1998), Chem. Biol. Interact. April24;111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

[0558] Polypeptides, including protein fusions to, or fragments thereof,of the present invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

[0559] In another embodiment, the invention provides a method ofdelivering compositions containing the polypeptides of the invention(e.g., compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

[0560] Polypeptides, protein fusions to, or fragments thereof, of thepresent invention are useful in enhancing the immunogenicity and/orantigenicity of proliferating cells or tissues, either directly, such aswould occur if the polypeptides of the present invention ‘vaccinated’the immune response to respond to proliferative antigens and immunogens,or indirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

[0561] Cardiovascular Disorders

[0562] Polynucleotides or polypeptides, or agonists or antagonists ofthe invention may be used to treat, prevent, and/or diagnosecardiovascular diseases, disorders, and/or conditions, includingperipheral artery disease, such as limb ischemia.

[0563] Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cor triatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

[0564] Cardiovascular diseases, disorders, and/or conditions alsoinclude heart disease, such as arrhythmias, carcinoid heart disease,high cardiac output, low cardiac output, cardiac tamponade, endocarditis(including bacterial), heart aneurysm, cardiac arrest, congestive heartfailure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema,heart hypertrophy, congestive cardiomyopathy, left ventricularhypertrophy, right ventricular hypertrophy, post-infarction heartrupture, ventricular septal rupture, heart valve diseases, myocardialdiseases, myocardial ischemia, pericardial effusion, pericarditis(including constrictive and tuberculous), pneumopericardium,postpericardiotomy syndrome, pulmonary heart disease, rheumatic heartdisease, ventricular dysfunction, hyperemia, cardiovascular pregnancycomplications, Scimitar Syndrome, cardiovascular syphilis, andcardiovascular tuberculosis.

[0565] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0566] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0567] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0568] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0569] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

[0570] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0571] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0572] Cerebrovascular diseases, disorders, and/or conditions includecarotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

[0573] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0574] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0575] Polynucleotides or polypeptides, or agonists or antagonists ofthe invention, are especially effective for the treatment of criticallimb ischemia and coronary disease.

[0576] Polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. Polypeptides of the invention may be administered aspart of a Therapeutic, described in more detail below. Methods ofdelivering polynucleotides of the invention are described in more detailherein.

[0577] Anti-Angiogenesis Activity

[0578] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye diseases, disorders, and/orconditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech.9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763(1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman,Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press,New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982);and Folkman et al., Science 221:719-725 (1983). In a number ofpathological conditions, the process of angiogenesis contributes to thedisease state. For example, significant data have accumulated whichsuggest that the growth of solid tumors is dependent on angiogenesis.Folkman and Klagsbrun, Science 235:442-447 (1987).

[0579] The present invention provides for treatment of diseases,disorders, and/or conditions associated with neovascularization byadministration of the polynucleotides and/or polypeptides of theinvention, as well as agonists or antagonists of the present invention.Malignant and metastatic conditions which can be treated with thepolynucleotides and polypeptides, or agonists or antagonists of theinvention include, but are not limited to, malignancies, solid tumors,and cancers described herein and otherwise known in the art (for areview of such disorders, see Fishman et al., Medicine, 2d Ed., J. B.Lippincott Co., Philadelphia (1985)). Thus, the present inventionprovides a method of treating, preventing, and/or diagnosing anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat or prevent a cancer or tumor. Cancers which may betreated, prevented, and/or diagnosed with polynucleotides, polypeptides,antagonists and/or agonists include, but are not limited to solidtumors, including prostate, lung, breast, ovarian, stomach, pancreas,larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum,cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primarytumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma;leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advancedmalignancies; and blood born tumors such as leukemias. For example,polynucleotides, polypeptides, antagonists and/or agonists may bedelivered topically, in order to treat or prevent cancers such as skincancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

[0580] Within yet other aspects, polynucleotides, polypeptides,antagonists and/or agonists may be utilized to treat superficial formsof bladder cancer by, for example, intravesical administration.Polynucleotides, polypeptides, antagonists and/or agonists may bedelivered directly into the tumor, or near the tumor site, via injectionor a catheter. Of course, as the artisan of ordinary skill willappreciate, the appropriate mode of administration will vary accordingto the cancer to be treated. Other modes of delivery are discussedherein.

[0581] Polynucleotides, polypeptides, antagonists and/or agonists may beuseful in treating, preventing, and/or diagnosing other diseases,disorders, and/or conditions, besides cancers, which involveangiogenesis. These diseases, disorders, and/or conditions include, butare not limited to: benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas;artheroscleric plaques; ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vesselgrowth) of the eye; rheumatoid arthritis; psoriasis; delayed woundhealing; endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

[0582] For example, within one aspect of the present invention methodsare provided for treating, preventing, and/or diagnosing hypertrophicscars and keloids, comprising the step of administering apolynucleotide, polypeptide, antagonist and/or agonist of the inventionto a hypertrophic scar or keloid.

[0583] Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating, preventing, and/ordiagnosing neovascular diseases of the eye, including for example,corneal neovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

[0584] Moreover, Ocular diseases, disorders, and/or conditionsassociated with neovascularization which can be treated, prevented,and/or diagnosed with the polynucleotides and polypeptides of thepresent invention (including agonists and/or antagonists) include, butare not limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

[0585] Thus, within one aspect of the present invention methods areprovided for treating or preventing neovascular diseases of the eye suchas corneal neovascularization (including corneal graftneovascularization), comprising the step of administering to a patient atherapeutically effective amount of a compound (as described above) tothe cornea, such that the formation of blood vessels is inhibited.Briefly, the cornea is a tissue which normally lacks blood vessels. Incertain pathological conditions however, capillaries may extend into thecornea from the pericorneal vascular plexus of the limbus. When thecornea becomes vascularized, it also becomes clouded, resulting in adecline in the patient's visual acuity. Visual loss may become completeif the cornea completely opacitates. A wide variety of diseases,disorders, and/or conditions can result in corneal neovascularization,including for example, corneal infections (e.g., trachoma, herpessimplex keratitis, leishmaniasis and onchocerciasis), immunologicalprocesses (e.g., graft rejection and Stevens-Johnson's syndrome), alkaliburns, trauma, inflammation (of any cause), toxic and nutritionaldeficiency states, and as a complication of wearing contact lenses.

[0586] Within particularly preferred embodiments of the invention, maybe prepared for topical administration in saline (combined with any ofthe preservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

[0587] Within other embodiments, the compounds described above may beinjected directly into the corneal stroma by an ophthalmologist undermicroscopic guidance. The preferred site of injection may vary with themorphology of the individual lesion, but the goal of the administrationwould be to place the composition at the advancing front of thevasculature (i.e., interspersed between the blood vessels and the normalcornea). In most cases this would involve perilimbic corneal injectionto “protect” the cornea from the advancing blood vessels. This methodmay also be utilized shortly after a corneal insult in order toprophylactically prevent corneal neovascularization. In this situationthe material could be injected in the perilimbic cornea interspersedbetween the corneal lesion and its undesired potential limbic bloodsupply. Such methods may also be utilized in a similar fashion toprevent capillary invasion of transplanted corneas. In asustained-release form injections might only be required 2-3 times peryear. A steroid could also be added to the injection solution to reduceinflammation resulting from the injection itself.

[0588] Within another aspect of the present invention, methods areprovided for treating or preventing neovascular glaucoma, comprising thestep of administering to a patient a therapeutically effective amount ofa polynucleotide, polypeptide, antagonist and/or agonist to the eye,such that the formation of blood vessels is inhibited. In oneembodiment, the compound may be administered topically to the eye inorder to treat or prevent early forms of neovascular glaucoma. Withinother embodiments, the compound may be implanted by injection into theregion of the anterior chamber angle. Within other embodiments, thecompound may also be placed in any location such that the compound iscontinuously released into the aqueous humor. Within another aspect ofthe present invention, methods are provided for treating or preventingproliferative diabetic retinopathy, comprising the step of administeringto a patient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eyes, such that theformation of blood vessels is inhibited.

[0589] Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

[0590] Within another aspect of the present invention, methods areprovided for treating or preventing retrolental fibroplasia, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eye, such that the formation of blood vessels is inhibited. Thecompound may be administered topically, via intravitreous injectionand/or via intraocular implants.

[0591] Additionally, diseases, disorders, and/or conditions which can betreated, prevented, and/or diagnosed with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

[0592] Moreover, diseases, disorders, and/or conditions and/or states,which can be treated, prevented, and/or diagnosed with thepolynucleotides, polypeptides, agonists and/or agonists include, but arenot limited to, solid tumors, blood born tumors such as leukemias, tumormetastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,rheumatoid arthritis, psoriasis, ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, and uvietis, delayed woundhealing, endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[0593] In one aspect of the birth control method, an amount of thecompound sufficient to block embryo implantation is administered beforeor after intercourse and fertilization have occurred, thus providing aneffective method of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

[0594] Polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may be incorporated into surgical sutures in order toprevent stitch granulomas.

[0595] Polynucleotides, polypeptides, agonists and/or agonists may beutilized in a wide variety of surgical procedures. For example, withinone aspect of the present invention a compositions (in the form of, forexample, a spray or film) may be utilized to coat or spray an area priorto removal of a tumor, in order to isolate normal surrounding tissuesfrom malignant tissue, and/or to prevent the spread of disease tosurrounding tissues. Within other aspects of the present invention,compositions (e.g., in the form of a spray) may be delivered viaendoscopic procedures in order to coat tumors, or inhibit angiogenesisin a desired locale. Within yet other aspects of the present invention,surgical meshes which have been coated with anti-angiogenic compositionsof the present invention may be utilized in any procedure wherein asurgical mesh might be utilized. For example, within one embodiment ofthe invention a surgical mesh laden with an anti-angiogenic compositionmay be utilized during abdominal cancer resection surgery (e.g.,subsequent to colon resection) in order to provide support to thestructure, and to release an amount of the anti-angiogenic factor.

[0596] Within further aspects of the present invention, methods areprovided for treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

[0597] Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

[0598] The polynucleotides, polypeptides, agonists and/or agonists ofthe present invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Surarnin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

[0599] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0600] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[0601] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

[0602] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem . . . 262(4): 1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

[0603] Diseases at the Cellular Level

[0604] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated, prevented, and/ordiagnosed by the polynucleotides or polypeptides and/or antagonists oragonists of the invention, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

[0605] Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented or diagnosed by thepolynucleotides or polypeptides, or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0606] Diseases associated with increased apoptosis that could betreated, prevented, and/or diagnosed by the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, includeAIDS; neurodegenerative diseases, disorders, and/or conditions (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune diseases, disorders, and/or conditions(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

[0607] Wound Healing and Epithelial Cell Proliferation

[0608] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe invention, may be clinically useful in stimulating wound healingincluding surgical wounds, excisional wounds, deep wounds involvingdamage of the dermis and epidermis, eye tissue wounds, dental tissuewounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitusulcers, arterial ulcers, venous stasis ulcers, burns resulting from heatexposure or chemicals, and other abnormal wound healing conditions suchas uremia, malnutrition, vitamin deficiencies and complicationsassociated with systemic treatment with steroids, radiation therapy andantineoplastic drugs and antimetabolites. Polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to promote dermal reestablishment subsequent to dermal loss

[0609] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to increase the adherence ofskin grafts to a wound bed and to stimulate re-epithelialization fromthe wound bed. The following are a non-exhaustive list of grafts thatpolynucleotides or polypeptides, agonists or antagonists of theinvention, could be used to increase adherence to a wound bed:autografts, artificial skin, allografts, autodermic graft, autoepidermicgrafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplasticgrafts, cutis graft, delayed graft, dermic graft, epidermic graft,fascia graft, full thickness graft, heterologous graft, xenograft,homologous graft, hyperplastic graft, lamellar graft, mesh graft,mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,pedicle graft, penetrating graft, split skin graft, thick split graft.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, can be used to promote skin strength and to improve theappearance of aged skin.

[0610] It is believed that the polynucleotides or polypeptides, and/oragonists or antagonists of the invention, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

[0611] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could also be used to reduce the sideeffects of gut toxicity that result from radiation, chemotherapytreatments or viral infections. The polynucleotides or polypeptides,and/or agonists or antagonists of the invention, may have acytoprotective effect on the small intestine mucosa. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, mayalso stimulate healing of mucositis (mouth ulcers) that result fromchemotherapy and viral infections.

[0612] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could further be used in full regenerationof skin in full and partial thickness skin defects, including burns,(i.e., repopulation of hair follicles, sweat glands, and sebaceousglands), treatment of other skin defects such as psoriasis. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could also be used to treat gastric anddoudenal ulcers and help heal by scar formation of the mucosal liningand regeneration of glandular mucosa and duodenal mucosal lining morerapidly. Inflamamatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to promote the resurfacing of the mucosalsurface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, isexpected to have a significant effect on the production of mucusthroughout the gastrointestinal tract and could be used to protect theintestinal mucosa from injurious substances that are ingested orfollowing surgery. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used to treat diseasesassociate with the under expression of the polynucleotides of theinvention.

[0613] Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated,prevented, and/or diagnosed using the polynucleotides or polypeptides,and/or agonists or antagonists of the invention. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

[0614] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

[0615] In addition, the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andII diabetes, where some islet cell function remains, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to maintain the islet function so as to alleviate, delay orprevent permanent manifestation of the disease. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used as an auxiliary in islet cell transplantationto improve or promote islet cell function.

[0616] Neurological Diseases

[0617] Nervous system diseases, disorders, and/or conditions, which canbe treated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB 12 deficiency, folic acid deficiency, Wernicke disease,tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primarydegeneration of the corpus callosum), and alcoholic cerebellardegeneration; (7) neurological lesions associated with systemic diseasesincluding, but not limited to, diabetes (diabetic neuropathy, Bell'spalsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8)lesions caused by toxic substances including alcohol, lead, orparticular neurotoxins; and (9) demyelinated lesions in which a portionof the nervous system is destroyed or injured by a demyelinating diseaseincluding, but not limited to, multiple sclerosis, humanimmunodeficiency virus-associated myelopathy, transverse myelopathy orvarious etiologies, progressive multifocal leukoencephalopathy, andcentral pontine myelinolysis.

[0618] In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

[0619] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

[0620] In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

[0621] Infectious Disease

[0622] A polypeptide or polynucleotide and/or agonist or antagonist ofthe present invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

[0623] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated, prevented, and/or diagnosed bya polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picomaviridae, Poxyiridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

[0624] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention include, but not limited to, include, but not limitedto, the following Gram-Negative and Gram-positive bacteria and bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis,Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis,Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis,Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli andEnterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella(e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

[0625] Moreover, parasitic agents causing disease or symptoms that canbe treated, prevented, and/or diagnosed by a polynucleotide orpolypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, the following families or class: Amebiasis,Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine,Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,Plasmodium virax, Plasmodium falciparium, Plasmodium malariae andPlasmodium ovale). These parasites can cause a variety of diseases orsymptoms, including, but not limited to: Scabies, Trombiculiasis, eyeinfections, intestinal disease (e.g., dysentery, giardiasis), liverdisease, lung disease, opportunistic infections (e.g., AIDS related),malaria, pregnancy complications, and toxoplasmosis. polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedto treat, prevent, and/or diagnose any of these symptoms or diseases. Inspecific embodiments, polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnosemalaria.

[0626] Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

[0627] Regeneration

[0628] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention can be used to differentiate, proliferate, andattract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997).) The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

[0629] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0630] Moreover, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase regeneration of tissuesdifficult to heal. For example, increased tendon/ligament regenerationwould quicken recovery time after damage. A polynucleotide orpolypeptide and/or agonist or antagonist of the present invention couldalso be used prophylactically in an effort to avoid damage. Specificdiseases that could be treated, prevented, and/or diagnosed include oftendinitis, carpal tunnel syndrome, and other tendon or ligamentdefects. A further example of tissue regeneration of non-healing woundsincludes pressure ulcers, ulcers associated with vascular insufficiency,surgical, and traumatic wounds.

[0631] Similarly, nerve and brain tissue could also be regenerated byusing a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention to proliferate and differentiate nerve cells.Diseases that could be treated, prevented, and/or diagnosed using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic diseases, disorders, and/orconditions (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated, prevented, and/or diagnosed using thepolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention.

[0632] Chemotaxis

[0633] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[0634] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treat, prevent,and/or diagnose inflammation, infection, hyperproliferative diseases,disorders, and/or conditions, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat, prevent,and/or diagnose wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treat,prevent, and/or diagnose wounds.

[0635] It is also contemplated that a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may inhibitchemotactic activity. These molecules could also be used to treat,prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention could be used as an inhibitor of chemotaxis.

[0636] Binding Activity

[0637] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

[0638] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[0639] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

[0640] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[0641] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[0642] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide level oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[0643] Additionally, the receptor to which a polypeptide of theinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

[0644] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0645] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0646] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[0647] Other preferred fragments are biologically active fragments ofthe polypeptides of the invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

[0648] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and 3[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0649] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messengerresponse is measured to determine if the compound is a potential agonistor antagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[0650] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

[0651] Also, one could identify molecules bind a polypeptide of theinvention experimentally by using the beta-pleated sheet regionscontained in the polypeptide sequence of the protein. Accordingly,specific embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, theamino acid sequence of each beta pleated sheet regions in a disclosedpolypeptide sequence. Additional embodiments of the invention aredirected to polynucleotides encoding polypeptides which comprise, oralternatively consist of, any combination or all of contained in thepolypeptide sequences of the invention. Additional preferred embodimentsof the invention are directed to polypeptides which comprise, oralternatively consist of, the amino acid sequence of each of the betapleated sheet regions in one of the polypeptide sequences of theinvention. Additional embodiments of the invention are directed topolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions in one of thepolypeptide sequences of the invention.

[0652] Targeted Delivery

[0653] In another embodiment, the invention provides a method ofdelivering compositions to targeted cells expressing a receptor for apolypeptide of the invention, or cells expressing a cell bound form of apolypeptide of the invention.

[0654] As discussed herein, polypeptides or antibodies of the inventionmay be associated with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions. In one embodiment, the invention provides amethod for the specific delivery of compositions of the invention tocells by administering polypeptides of the invention (includingantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[0655] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

[0656] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[0657] Drug Screening

[0658] Further contemplated is the use of the polypeptides of thepresent invention, or the polynucleotides encoding these polypeptides,to screen for molecules which modify the activities of the polypeptidesof the present invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

[0659] This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

[0660] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thepolypeptides of the present invention. These methods comprise contactingsuch an agent with a polypeptide of the present invention or a fragmentthereof and assaying for the presence of a complex between the agent andthe polypeptide or a fragment thereof, by methods well known in the art.In such a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

[0661] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to thepolypeptides of the present invention, and is described in great detailin European Patent Application 84/03564, published on Sep. 13, 1984,which is incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with polypeptides of the present inventionand washed. Bound polypeptides are then detected by methods well knownin the art. Purified polypeptides are coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

[0662] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptides of the present invention specifically compete with a testcompound for binding to the polypeptides or fragments thereof. In thismanner, the antibodies are used to detect the presence of any peptidewhich shares one or more antigenic epitopes with a polypeptide of theinvention.

[0663] The human HLRRSI1 polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a HLRRSI1polypeptide, or a bindable peptide fragment, of this invention,comprising providing a plurality of compounds, combining the HLRRSI1polypeptide, or a bindable peptide fragment, with each of a plurality ofcompounds for a time sufficient to allow binding under suitableconditions and detecting binding of the HLRRSI1 polypeptide or peptideto each of the plurality of test compounds, thereby identifying thecompounds that specifically bind to the HLRRSI1 polypeptide or peptide.

[0664] Methods of identifying compounds that modulate the activity ofthe novel human HLRRSI1 polypeptides and/or peptides are provided by thepresent invention and comprise combining a potential or candidatecompound or drug modulator of leucine-rich repeat protein biologicalactivity with an HLRRSI1 polypeptide or peptide, for example, theHLRRSI1 amino acid sequence as set forth in SEQ ID NOS:2, and measuringan effect of the candidate compound or drug modulator on the biologicalactivity of the HLRRSI1 polypeptide or peptide. Such measurable effectsinclude, for example, physical binding interaction; the ability tocleave a suitable leucine-rich repeat protein substrate; effects onnative and cloned HLRRSI1-expressing cell line; and effects ofmodulators or other leucine-rich repeat protein-mediated physiologicalmeasures.

[0665] Another method of identifying compounds that modulate thebiological activity of the novel HLRRSI1 polypeptides of the presentinvention comprises combining a potential or candidate compound or drugmodulator of a leucine-rich repeat protein biological activity with ahost cell that expresses the HLRRSI1 polypeptide and measuring an effectof the candidate compound or drug modulator on the biological activityof the HLRRSI1 polypeptide. The host cell can also be capable of beinginduced to express the HLRRSI1 polypeptide, e.g., via inducibleexpression. Physiological effects of a given modulator candidate on theHLRRSI1 polypeptide can also be measured. Thus, cellular assays forparticular leucine-rich repeat protein modulators may be either directmeasurement or quantification of the physical biological activity of theHLRRSI1 polypeptide, or they may be measurement or quantification of aphysiological effect. Such methods preferably employ a HLRRSI1polypeptide as described herein, or an overexpressed recombinant HLRRSI1polypeptide in suitable host cells containing an expression vector asdescribed herein, wherein the HLRRSI1 polypeptide is expressed,overexpressed, or undergoes upregulated expression.

[0666] Another aspect of the present invention embraces a method ofscreening for a compound that is capable of modulating the biologicalactivity of a HLRRSI1 polypeptide, comprising providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a HLRRSI1 polypeptide, or a functional peptide or portionthereof (e.g., SEQ ID NOS:2); determining the biological activity of theexpressed HLRRSI1 polypeptide in the absence of a modulator compound;contacting the cell with the modulator compound and determining thebiological activity of the expressed HLRRSI1 polypeptide in the presenceof the modulator compound. In such a method, a difference between theactivity of the HLRRSI1 polypeptide in the presence of the modulatorcompound and in the absence of the modulator compound indicates amodulating effect of the compound.

[0667] Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as leucine-rich repeat protein modulators can be anysmall chemical compound, or biological entity (e.g., protein, sugar,nucleic acid, lipid). Test compounds will typically be small chemicalmolecules and peptides. Generally, the compounds used as potentialmodulators can be dissolved in aqueous or organic (e.g., DMSO-based)solutions. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource. Assays are typically run in parallel, for example, in microtiterformats on microtiter plates in robotic assays. There are many suppliersof chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

[0668] High throughput screening methodologies are particularlyenvisioned for the detection of modulators of the novel HLRRSI1polynucleotides and polypeptides described herein. Such high throughputscreening methods typically involve providing a combinatorial chemicalor peptide library containing a large number of potential therapeuticcompounds (e.g., ligand or modulator compounds). Such combinatorialchemical libraries or ligand libraries are then screened in one or moreassays to identify those library members (e.g., particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds so identified can serve as conventional lead compounds, orcan themselves be used as potential or actual therapeutics.

[0669] A combinatorial chemical library is a collection of diversechemical compounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

[0670] The preparation and screening of combinatorial chemical librariesis well known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptoids(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

[0671] Devices for the preparation of combinatorial libraries arecommercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A AppliedBiosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.).In addition, a large number of combinatorial libraries are commerciallyavailable (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia;Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and thelike).

[0672] In one embodiment, the invention provides solid phase based invitro assays in a high throughput format, where the cell or tissueexpressing an ion channel is attached to a solid phase substrate. Insuch high throughput assays, it is possible to screen up to severalthousand different modulators or ligands in a single day. In particular,each well of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

[0673] In another of its aspects, the present invention encompassesscreening and small molecule (e.g., drug) detection assays which involvethe detection or identification of small molecules that can bind to agiven protein, i.e., a HLRRSI1 polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

[0674] In such binding-based detection, identification, or screeningassays, a functional assay is not typically required. All that is neededis a target protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

[0675] An example of such an assay is the fluorescence based thermalshift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

[0676] To purify a HLRRSI1 polypeptide or peptide to measure abiological binding or ligand binding activity, the source may be a wholecell lysate that can be prepared by successive freeze-thaw cycles (e.g.,one to three) in the presence of standard protease inhibitors. TheHLRRSI1 polypeptide may be partially or completely purified by standardprotein purification methods, e.g., affinity chromatography usingspecific antibody described infra, or by ligands specific for an epitopetag engineered into the recombinant HLRRSI1 polypeptide molecule, alsoas described herein. Binding activity can then be measured as described.

[0677] Compounds which are identified according to the methods providedherein, and which modulate or regulate the biological activity orphysiology of the HLRRSI1 polypeptides according to the presentinvention are a preferred embodiment of this invention. It iscontemplated that such modulatory compounds may be employed in treatmentand therapeutic methods for treating a condition that is mediated by thenovel HLRRSI1 polypeptides by administering to an individual in need ofsuch treatment a therapeutically effective amount of the compoundidentified by the methods described herein.

[0678] In addition, the present invention provides methods for treatingan individual in need of such treatment for a disease, disorder, orcondition that is mediated by the HLRRSI1 polypeptides of the invention,comprising administering to the individual a therapeutically effectiveamount of the HLRRSI1-modulating compound identified by a methodprovided herein.

[0679] Antisense and Ribozyme (Antagonists)

[0680] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:1, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

[0681] For example, the use of c-myc and c-myb antisense RNA constructsto inhibit the growth of the non-lymphocytic leukemia cell line HL-60and other cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcOR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcOR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

[0682] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0683] In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

[0684] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0685] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

[0686] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al.,BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g.,Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotidemay be conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

[0687] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0688] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0689] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0690] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[0691] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

[0692] While antisense nucleotides complementary to the coding regionsequence of the invention could be used, those complementary to thetranscribed untranslated region are most preferred.

[0693] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science,247:1222-1225 (1990). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[0694] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[0695] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[0696] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[0697] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[0698] The antagonist/agonist may also be employed to treat, prevent,and/or diagnose the diseases described herein.

[0699] Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

[0700] invention, and/or (b) a ribozyme directed to the polynucleotideof the present invention.

[0701] Biotic Associations

[0702] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability, eitherdirectly or indirectly, to initiate and/or maintain biotic associationswith other organisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

[0703] The mechanism by which a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may increase the hostorganisms ability, either directly or indirectly, to initiate and/ormaintain biotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

[0704] In an alternative embodiment, a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may decrease thehost organisms ability to form biotic associations with anotherorganism, either directly or indirectly. The mechanism by which apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may decrease the host organisms ability, eitherdirectly or indirectly, to initiate and/or maintain biotic associationswith another organism is variable, though may include, modulatingosmolarity to undesirable levels, modulating pH to undesirable levels,modulating secretions of organic acids, modulating the secretion ofspecific proteins, phenolic compounds, nutrients, or the decreasedexpression of a protein required for host-biotic organisms interactions(e.g., a receptor, ligand, etc.). Additional mechanisms are known in theart and are encompassed by the invention (see, for example, “MicrobialSignalling and Communication”, eds., R. England, G. Hobbs, N. Bainton,and D. McL. Roberts, Cambridge University Press, Cambridge, (1999);which is hereby incorporated herein by reference).

[0705] The hosts ability to maintain biotic associations with aparticular pathogen has significant implications for the overall healthand fitness of the host. For example, human hosts have symbiosis withenteric bacteria in their gastrointestinal tracts, particularly in thesmall and large intestine. In fact, bacteria counts in feces of thedistal colon often approach 10¹² per milliliter of feces. Examples ofbowel flora in the gastrointestinal tract are members of theEnterobacteriaceae, Bacteriodes, in addition to a-hemolyticstreptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria,Costridia, lactobacilli, and yeasts. Such bacteria, among other things,assist the host in the assimilation of nutrients by breaking down foodstuffs not typically broken down by the hosts digestive system,particularly in the hosts bowel. Therefore, increasing the hosts abilityto maintain such a biotic association would help assure proper nutritionfor the host.

[0706] Aberrations in the enteric bacterial population of mammals,particularly humans, has been associated with the following disorders:diarrhea, ileus, chronic inflammatory disease, bowel obstruction,duodenal diverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

[0707] The composition of the intestinal flora, for example, is basedupon a variety of factors, which include, but are not limited to, theage, race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

[0708] Although the predominate intestinal flora comprises anaerobicorganisms, an underlying percentage represents aerobes (e.g., E. coli).This is significant as such aerobes rapidly become the predominateorganisms in intraabdominal infections—effectively becomingopportunistic early in infection pathogenesis. As a result, there is anintrinsic need to control aerobe populations, particularly for immunecompromised individuals.

[0709] In a preferred embodiment, a polynucleotides and polypeptides,including agonists, antagonists, and fragments thereof, are useful forinhibiting biotic associations with specific enteric symbiont organismsin an effort to control the population of such organisms.

[0710] Biotic associations occur not only in the gastrointestinal tract,but also on an in the integument. As opposed to the gastrointestinalflora, the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M. sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P. acnes), gram-negative bacilli (e.g.,Acinebacter species), and fungi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

[0711] Aberrations in the cutaneous flora are associated with a numberof significant diseases and/or disorders, which include, but are notlimited to the following: impetigo, ecthyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

[0712] Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.,Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

[0713] Pheromones

[0714] In another embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may increase theorganisms ability to synthesize and/or release a pheromone. Such apheromone may, for example, alter the organisms behavior and/ormetabolism.

[0715] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones. Preferably, any of the pheromones, and/or volatilesreleased from the organism, or induced, by a polynucleotide orpolypeptide and/or agonist or antagonist of the invention havebehavioral effects the organism.

[0716] Other Activities

[0717] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration, as discussed above.

[0718] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0719] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat, prevent, and/or diagnoseneuronal damage which occurs in certain neuronal disorders orneuro-degenerative conditions such as Alzheimer's disease, Parkinson'sdisease, and AIDS-related complex. The polypeptide of the invention mayhave the ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

[0720] The polypeptide of the present invention may be also be employedto prevent skin aging due to sunburn by stimulating keratinocyte growth.

[0721] The polypeptide of the invention may also be employed forpreventing hair loss, since FGF family members activate hair-formingcells and promotes melanocyte growth. Along the same lines, thepolypeptides of the present invention may be employed to stimulategrowth and differentiation of hematopoietic cells and bone marrow cellswhen used in combination with other cytokines.

[0722] The polypeptide of the invention may also be employed to maintainorgans before transplantation or for supporting cell culture of primarytissues.

[0723] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[0724] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also increase or decrease thedifferentiation or proliferation of embryonic stem cells, besides, asdiscussed above, hematopoietic lineage.

[0725] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

[0726] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

[0727] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used as a food additive orpreservative, such as to increase or decrease storage capabilities, fatcontent, lipid, protein, carbohydrate, vitamins, minerals, cofactors orother nutritional components.

[0728] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

[0729] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

[0730] Other Preferred Embodiments

[0731] Other preferred embodiments of the claimed invention include anisolated nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least about 50 contiguousnucleotides in the nucleotide sequence of SEQ ID NO:1 wherein X is anyinteger as defined in Table I.

[0732] Also preferred is a nucleic acid molecule wherein said sequenceof contiguous nucleotides is included in the nucleotide sequence of SEQID NO:1 in the range of positions beginning with the nucleotide at aboutthe position of the “5′ NT of Start Codon of ORF” and ending with thenucleotide at about the position of the “3′ NT of ORF” as defined forSEQ ID NO:1 in Table I.

[0733] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:1.

[0734] Further preferred is an isolated nucleic acid molecule comprisinga nucleotide sequence which is at least 95% identical to a sequence ofat least about 500 contiguous nucleotides in the nucleotide sequence ofSEQ ID NO:1.

[0735] A further preferred embodiment is a nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thenucleotide sequence of SEQ ID NO:1 beginning with the nucleotide atabout the position of the “5′ NT of ORF” and ending with the nucleotideat about the position of the “3′ NT of ORF” as defined for SEQ ID NO:1in Table I.

[0736] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence of SEQ ID NO:1.

[0737] Also preferred is an isolated nucleic acid molecule whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule, wherein said nucleic acid molecule which hybridizes does nothybridize under stringent hybridization conditions to a nucleic acidmolecule having a nucleotide sequence consisting of only A residues orof only T residues.

[0738] Also preferred is a composition of matter comprising a DNAmolecule which comprises a cDNA clone identified by a cDNA CloneIdentifier in Table I, which DNA molecule is contained in the materialdeposited with the American Type Culture Collection and given the ATCCDeposit Number shown in Table I for said cDNA Clone Identifier.

[0739] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of a cDNAclone identified by a cDNA Clone Identifier in Table I, which DNAmolecule is contained in the deposit given the ATCC Deposit Number shownin Table I.

[0740] Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of the complete open reading frame sequence encodedby said cDNA clone.

[0741] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bysaid cDNA clone.

[0742] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to sequence of at least 500 contiguous nucleotides in thenucleotide sequence encoded by said cDNA clone.

[0743] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence encoded by said cDNAclone.

[0744] A further preferred embodiment is a method for detecting in abiological sample a nucleic acid molecule comprising a nucleotidesequence which is at least 95% identical to a sequence of at least 50contiguous nucleotides in a sequence selected from the group consistingof: a nucleotide sequence of SEQ ID NO:1 wherein X is any integer asdefined in Table I; and a nucleotide sequence encoded by a cDNA cloneidentified by a cDNA Clone Identifier in Table I and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableI; which method comprises a step of comparing a nucleotide sequence ofat least one nucleic acid molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidnucleic acid molecule in said sample is at least 95% identical to saidselected sequence.

[0745] Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

[0746] A further preferred embodiment is a method for identifying thespecies, tissue or cell type of a biological sample which methodcomprises a step of detecting nucleic acid molecules in said sample, ifany, comprising a nucleotide sequence that is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:1wherein X is any integer as defined in Table I; and a nucleotidesequence encoded by a cDNA clone identified by a cDNA Clone Identifierin Table I and contained in the deposit with the ATCC Deposit Numbershown for said cDNA clone in Table I.

[0747] The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

[0748] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene encoding a protein identified in Table I, which methodcomprises a step of detecting in a biological sample obtained from saidsubject nucleic acid molecules, if any, comprising a nucleotide sequencethat is at least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:1 wherein X is any integer as definedin Table I; and a nucleotide sequence encoded by a cDNA clone identifiedby a cDNA Clone Identifier in Table I and contained in the deposit withthe ATCC Deposit Number shown for said cDNA clone in Table I.

[0749] The method for diagnosing a pathological condition can comprise astep of detecting nucleic acid molecules comprising a nucleotidesequence in a panel of at least two nucleotide sequences, wherein atleast one sequence in said panel is at least 95% identical to a sequenceof at least 50 contiguous nucleotides in a sequence selected from saidgroup.

[0750] Also preferred is a composition of matter comprising isolatednucleic acid molecules wherein the nucleotide sequences of said nucleicacid molecules comprise a panel of at least two nucleotide sequences,wherein at least one sequence in said panel is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:1wherein X is any integer as defined in Table I; and a nucleotidesequence encoded by a cDNA clone identified by a cDNA Clone Identifierin Table I and contained in the deposit with the ATCC Deposit Numbershown for said cDNA clone in Table I. The nucleic acid molecules cancomprise DNA molecules or RNA molecules.

[0751] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID NO:2 whereinY is any integer as defined in Table I.

[0752] Also preferred is a polypeptide, wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of SEQ IDNO:2 in the range of positions “Total AA of the Open Reading Frame(ORF)” as set forth for SEQ ID NO:2 in Table I.

[0753] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:2.

[0754] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:2.

[0755] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the complete amino acid sequenceof SEQ ID NO:2.

[0756] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of a proteinencoded by a cDNA clone identified by a cDNA Clone Identifier in Table Iand contained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

[0757] Also preferred is a polypeptide wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of theprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

[0758] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of the protein encodedby a cDNA clone identified by a cDNA Clone Identifier in Table I andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

[0759] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the protein encodedby a cDNA clone identified by a cDNA Clone Identifier in Table I andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table I.

[0760] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the amino acid sequence of theprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

[0761] Further preferred is an isolated antibody which bindsspecifically to a polypeptide comprising an amino acid sequence that isat least 90% identical to a sequence of at least 10 contiguous aminoacids in a sequence selected from the group consisting of: an amino acidsequence of SEQ ID NO:2 wherein Y is any integer as defined in Table I;and a complete amino acid sequence of a protein encoded by a cDNA cloneidentified by a cDNA Clone Identifier in Table I and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableI.

[0762] Further preferred is a method for detecting in a biologicalsample a polypeptide comprising an amino acid sequence which is at least90% identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:2 wherein Y is any integer as defined in Table I; and acomplete amino acid sequence of a protein encoded by a cDNA cloneidentified by a cDNA Clone Identifier in Table I and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableI; which method comprises a step of comparing an amino acid sequence ofat least one polypeptide molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidpolypeptide molecule in said sample is at least 90% identical to saidsequence of at least 10 contiguous amino acids.

[0763] Also preferred is the above method wherein said step of comparingan amino acid sequence of at least one polypeptide molecule in saidsample with a sequence selected from said group comprises determiningthe extent of specific binding of polypeptides in said sample to anantibody which binds specifically to a polypeptide comprising an aminoacid sequence that is at least 90% identical to a sequence of at least10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is anyinteger as defined in Table I; and a complete amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

[0764] Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

[0765] Also preferred is a method for identifying the species, tissue orcell type of a biological sample which method comprises a step ofdetecting polypeptide molecules in said sample, if any, comprising anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is anyinteger as defined in Table I; and a complete amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

[0766] Also preferred is the above method for identifying the species,tissue or cell type of a biological sample, which method comprises astep of detecting polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from theabove group.

[0767] Also preferred is a method for diagnosing a pathologicalcondition associated with an organism with abnormal structure orexpression of a gene encoding a protein identified in Table I, whichmethod comprises a step of detecting in a biological sample obtainedfrom said subject polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from thegroup consisting of: an amino acid sequence of SEQ ID NO:2 wherein Y isany integer as defined in Table I; and a complete amino acid sequence ofa protein encoded by a cDNA clone identified by a cDNA Clone Identifierin Table I and contained in the deposit with the ATCC Deposit Numbershown for said cDNA clone in Table I.

[0768] In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

[0769] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is anyinteger as defined in Table I; and a complete amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

[0770] Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

[0771] Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is anyinteger as defined in Table I; and a complete amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I.

[0772] Further preferred is a method of making a recombinant vectorcomprising inserting any of the above isolated nucleic acid molecule(s)into a vector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector into a host cell, as well as therecombinant host cell produced by this method.

[0773] Also preferred is a method of making an isolated polypeptidecomprising culturing this recombinant host cell under conditions suchthat said polypeptide is expressed and recovering said polypeptide. Alsopreferred is this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is aprotein comprising an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:2 wherein Y is aninteger set forth in Table I and said position of the “Total AA of ORF”of SEQ ID NO:2 is defined in Table I; and an amino acid sequence of aprotein encoded by a cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table I. The isolated polypeptide produced bythis method is also preferred.

[0774] Also preferred is a method of treatment of an individual in needof an increased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

[0775] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

REFERENCES

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[0777] K Hofmann, W Stoffel. TMbase—A database of membrane spanningproteins segments. Biol. Chem. Hoppe-Seyler 347:166, 1993.

[0778] Bertin, J., and DiStefano, P. S. (2000). The PYRIN Domain: Anovel Motif found in apoptosis and inflammation proteins. Cell DeathDiffer. In press.

[0779] Dixon, M. S., Golstein, C., Thomas, C. M., van der Biezen, E. A.,and Jones, J. D. G. (2000). Genetic complexity of pathogen perception byplants: The example of Rcr3, a tomato gene required specifically byCf-2. Proc. Natl. Acad. Sci. USA 97, 8807-8814.

[0780] Eldon, E., Kooyer, S., D'Evelyn, D., Duman, M., Lawinger, P.,Botas, J., and Bellen, H. (1994). The Drosophila 18 wheeler is requiredfor morphogenesis and has striking similarities to Toll. Development120, 885-899.

[0781] Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992).Identification of programmed cell death in situ via specific labeling ofnuclear DNA fragmentation. J. Cell Biol. 119, 493-501.

[0782] Halfon, M., Hashimoto, C., and Keshishian, H. (1995). TheDrosophila Toll gene functions zygotically and is necessary for propermotoneuron and muscle development. Dev. Biol. 169, 151-167.

[0783] Harton, J. A., and Ting, J. P. (2000). Class II transactivator:Mastering the art of major histocompatibility complex expression.Molecular and Cellular Biology 20, 6185-6194.

[0784] Inohara, N., Koseki, T., Lin, J., Peso, L., Lucas, P. C., Chen,F. F., Ogura, Y., and Nunez, G. (2000). An induced proximity model forNF-kB activation in the Nod 1/RICK and RIP signaling pathways. Journalof Biological Chemistry 275, 27823-27831.

[0785] Inohara, N., Koseki, T., Peso, L., Hu, Y., Yee, C., Chen, S.,Carrio, R., Merina, J., Liu, D., Ni, J., and Nunez, G. (1999). Nod1, anApaf-1-like activator of caspase-9 and nuclear factor-kB. Journal ofBiological Chemistry 274, 14560-14567.

[0786] Jacobs, J., and Goodman, C. (1989). Embryonic development of axonpathways in the Drosophila CNS. I. A glial cell scaffold appears beforethe first growth cones. J. Neurosci. 9, 2402-2411.

[0787] Jones, D. A., McIntire, L. V., Smith, C. W., and Picker, L. J.(1994). A two step adhesion cascade for T-cell/endothelial cellinteraction under flow conditions. J. Clin. Invest. 94, 2443-2450.

[0788] Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M.,Margolis, R. K., and Margolis, R. U. (1999). Mammalina homologues of theDrosophila slit protein are ligands of the herparan sulfateproteoglycan-i in brain. J. Biol. Chem . . . 274, 17885-1792.

[0789] Schneider, D. S., Hudson, K. L., Lin, T., and Anderson, K. V.(1991). Dominant and recessive mutations define functional domains ofToll, a transmembrane protein required for dorsal-ventral polarity inthe Drosophila embryo. Genes and Development 5, 797-807.

[0790] Sean, G. S., Buchanan, C., and Gay, N. J. (1996). Structural andfunctional diversity in the leucine rich repeat family of proteins.Prog. Biophys. Molec. Biol. 65, 1-44.

[0791] Tong, Z. B., and Nelson, L. M. (1999). A mouse gene encoding anoocyte antigen associated with autoimmune premature ovarian failure.Endocrinology 140, 3720-3726.

[0792] van Der Voort, R., Keehnen, R. M., Beuling, E. A., Spaargaren,M., and Pals, S. T. (2000). Regulation of cytokine signaling by B cellantigen receptor and CD40-controlled expression of heparan sulfateproteoglycans. J. Exp. Med. 192, 1115-1124.

[0793] Verbeek, M. M., Otte-Holler, I., van den Born, J., van denHeuvel, L. P., Wesseling, P., and M., d. W. R. (1999). Agrin is a majorheparan sulfate proteoglycan accummulating in Alzheimer's disease brain.Am. J. Pathol. 155, 2115-2125.

EXAMPLES Description of the Preferred Embodiments Example 1Bioinformatics Analysis

[0794] A Leucine-rich repeat (LLR) domain containing protein, annotatedas Angiotensin/Vasopressin receptor AII/AVP (Genbank Accession:AAC39910; SEQ ID NO:25) and other LLR-domain containing proteins such asKIAA0926 (Genbank Accession: NP_(—)055737; SEQ ID NO:26) were used asprobes to search the EST databases from Incyte and the public domain, inaddition to the genomic database from the Human Genome Project. Thesearch program used was BLAST (Basic Local Alignment Search Tool). Fromthis analysis, ESTs and exons encoding potential novel candidates,related to Angiotensin/Vasopressin receptor, were identified based onsequence homology. The potential candidates (Incyte ESTs: 1632960H1 andPublic domain EST GI number: g201045) were sequenced. Two clones wereidentified, entitled SILRR1A (SEQ ID NO:5; ATCC Deposit No. PTA-2679)and SILRR1B (SEQ ID NO:6; ATCC Deposit No. PTA-2674) was obtained usingthe EST sequence information. The sequence of these two clones wascombined through contig analysis procedures known in the art to obtainthe full-length clone encoding the novel HLRRSI-1 protein. The completeprotein sequences of these proteins were analyzed for potentialtransmembrane domains. TMPRED program (5) was used for transmembraneprediction. Also, these proteins were analyzed for potential motifs andprotein domains. Motifs program in GCG (GCG is a software package fromGenetics Computer Group of Wisconsin) was used for identifying thepotential motifs in the protein. Protein domains were analyzed by usingHMMER. HMMER is a freely distributable implementation of profile HiddenMarkov Model (HMM) software for protein sequence analysis(http://hmmer.wustl.edu/). The protein domain search set used was Pfam(http://pfam.wustl.edu/). Pfam is a large collection of multiplesequence alignments and hidden Markov models of protein domains covering2478 protein families. By these analyses, the HLRRSI1 protein has beenpredicted to comprise one or more leucine-rich repeat domains.

Example 2 Method for Constructing a Size Fractionated Brain and TestisCdna Library

[0795] Brain and testis poly A+ RNA was purchased from Clontech andconverted into double stranded cDNA using the SuperScript™ PlasmidSystem for cDNA Synthesis and Plasmid Cloning (Life Technologies) exceptthat no radioisotope was incorporated in either of the cDNA synthesissteps and that the cDNA was fractionated by HPLC. This was accomplishedon a TransGenomics HPLC system equipped with a size exclusion column(TosoHass) with dimensions of 7.8 mm×30 cm and a particle size of 10 μm.Tris buffered saline was used as the mobile phase, and the column wasrun at a flow rate of 0.5 mL/min. The resulting chromatograms wereanalyzed to determine which fractions should be pooled to obtain thelargest cDNA's; generally fractions that eluted in the range of 12 to 15minutes were pooled. The cDNA was precipitated prior to ligation intothe Sal I/Not I sites in the pSport vector supplied with the kit. Usinga combination of PCR with primers directed to the ends of the vector andSal I/Not I restriction enzyme digestion of mini-prep DNA, it wasdetermined that the average insert size of the library was greater the3.5 Kb. The overall complexity of the library was greater than 10⁷independent clones. The library was amplified in semi-solid agar for 2days at 30° C.

[0796] An aliquot (200 microliters) of the amplified library wasinoculated into a 200 ml culture for single-stranded DNA isolation bysuper-infection with a f1 helper phage. After overnight grow, thereleased phage particles with precipitated with PEG and the DNA isolatedwith proteinase K, SDS and phenol extractions. The single strandedcircular DNA was concentrated by ethanol precipitation and used for thecDNA capture experiments described below.

Example 3 Cloning of the Novel Human HLRRSI1 Polynucleotide

[0797] Using the following EST sequence, the following PCR primer pairsand antisense 80 bp 5′ biotinylated oligonucleotides (shown in TableIII) were designed and obtained from Genset Oligos (San Diego, Calif.)for use in the cloning methods described below.

[0798] BMS Clone #12

[0799] INCYTE 1632960H1

[0800] EST Sequence: GCCACTTGGTGCTCACCACGCGCTTCCTCTTCGGACT (SEQ ID NO:7)GCTGAGCGCGGAGGGATGCGCGACATCGAGCGCCACTTCGGCTGCATGGTTTCAGAGCGTGTGAAGCAGGAGGCCCTGCGGTGGGTGCAGGGACAGGGACAGGGCTGCCCCGGAGTGGCACCAGAGGTGACCGAGGGGGCCAAAGGGCTCGAGGACACCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCAACTACCCACTGGAGTTGCTGTACTGCCTGTACGAGACGCAGGAGGACGCGTTTGTNCGCCAAA GCCCTGTGCCGGTTCCCG

[0801] TABLE III Oligonucleotides. Oligo Oligo Oligonucleotide SEQ IDNo. Name Sequence Length NO: 363 GPCR-12s CATGGTTTCAGAGCGTGTGAA 21 11364 GPCR-12a TCGTACAGGCAGTACAGCAAC 23 12 TC 596 GPCR-12CTTCACACGCTCTGAAACCAT 80 13 GCAGCCGAAGTGGCGCTCGAT GTCGCGCATCCCTCCGCGCTCAGCAGTCCGAAGAGGAA

[0802] One microliter (one hundred and fifty nanograms) of abiotinylated oligo (SEQ ID NO:13) was added to six microliters (sixmicrograms) of a mixture of the single-stranded covalently closedcircular brain and testis cDNA libraries described herein, and sevenmicroliters of 100% formamide in a 0.5 ml PCR tube. The mixture washeated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA librarymixture and incubated at 42° C. for 26 hours. Hybrids between thebiotinylated oligo and the circular cDNA were isolated by diluting thehybridization mixture to 220 microliters in a solution containing 1 MNaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125microliters of streptavidin magnetic beads. This solution was incubatedat 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. Thebeads were separated from the solution with a magnet and the beadswashed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

[0803] The single stranded cDNAs were released from the biotinlyatedoligo/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 mins. Six microlitersof 3 M Sodium Acetate was added along with 15 micrograms of glycogen andthe solution ethanol precipitated with 120 microliters of 100% ethanol.The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0),1 mM EDTA, pH 8.0). The single stranded cDNA was converted into doublestrands in a thermal cycler by mixing 5 microliters of the captured DNAwith 1.5 microliters 10 micromolar standard SP6 primer (homologous to asequence on the cDNA cloning vector) and 1.5 microliters of 10×PCRbuffer. The mixture was heated to 95° C. for 20 seconds, then rampeddown to 59° C. At this time 15 microliters of a repair mix, that waspreheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs(1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters ofwater, and 0.25 microliters of Taq polymerase). The solution was rampedback to 73° C. and incubated for 23 mins. The repaired DNA was ethanolprecipitate and resuspended in 10 microliters of TE. Two microliterswere electroporated in E. coli DH12S cells and resulting colonies werescreen by PCR, using a primer pairs designed from the EST sequences toidentify the proper cDNAs. Those cDNA clones that were positive by PCRhad the inserts sized and two clones for each probe were chosen for DNAsequencing.

[0804] The full-length nucleotide sequence and the encoded polypeptidefor HLRRSI1 is shown in FIGS. 1A-C.

Example 4 Expression Profiling of the Novel Human HLRRSI1 Polypeptide

[0805] The following PCR primer pair was used to measure the steadystate levels of HLRRSI1 mRNA by quantitative PCR: Sense:5′-CATGGTTTCAGAGCGTGTGAA-3′ (SEQ ID NO:11) Antisense:5′-TCGTACAGGCAGTACAGCAACTC-3′ (SEQ ID NO:12)

[0806] Briefly, first strand cDNA was made from commercially availablemRNA (Clontech) and subjected to real time quantitative PCR using a PE5700 instrument (Applied Biosystems, Foster City, Calif.) which detectsthe amount of DNA amplified during each cycle by the fluorescent outputof SYBR green, a DNA binding dye specific for double strands. The primerpairs provided above were used in the PCR reaction. The specificity ofthe primer pair for its target was verified by performing a thermaldenaturation profile at the end of the run which gave an indication ofthe number of different DNA hybridization complexes present bydetermining melting Tm. In the case of the novel HLRRSI1 gene primerpairs, only one DNA fragment was detected having a homogeneous meltingpoint. Contributions of contaminating genomic DNA to the assessment oftissue abundance is controlled by performing the PCR with first strandcDNA made with and without reverse transcriptase. In all cases, thecontribution of material amplified in the no reverse transcriptasecontrols was negligible.

[0807] Small variations in the amount of cDNA used in each tube wasdetermined by performing a parallel experiment using a primer pair for agene expressed in equal amounts in all tissues, cyclophilin. These datawere used to normalize the data obtained with the HLRRSI1 primer pairsdescribed herein. The PCR data was converted into a relative assessmentof the difference in transcript abundance amongst the tissues tested andthe data are presented in bar graph form in FIG. 4. As shown,transcripts corresponding to HLRRSI1 were expressed highly in the smallintestine, and to a lesser extent, in liver, spleen, and lymph node.

Example 5 Method of Assessing the Ability of HLRRSI1 to ModulateApoptosis

[0808] The role of the novel HLRRSI1 polypeptides in either promoting orinhibiting apoptosis could be determined by the generation oftransfected cell lines with the HLRRSI1 polynucleotides of the presentinvention, either transient or stable, using methods known in the artand/or described herein, and any combination of commonly used assays forthe detection of DNA fragmentation. One representative example being theTUNEL assay (Gavrieli, Y., Sherman, Y., Ben, Sasson, SA, J. Cell, Biol.,119(3):493-501, (1992); which is hereby incorporated herein by referencein its entirety) which involves end labeling broken ends ofdouble-stranded DNA with biotin-conjugated dUTP using terminaltransferase. Cells undergoing cell death can then be easily detected bystaining with FITC-conjugated streptavidin and flow cytometricquantitation.

[0809] Alternatively, HLRRSI1 can be expressed by transforming amammalian cell line such as COS7, HeLa or CHO with an eukaryoticexpression vector encoding HLRRSI1. Eukaryotic expression vectors arecommercially available, and the techniques to introduce them into cellsare well known to those skilled in the art. The cells with and withoutthe HLRRSI1 expression vector are incubated for 48-72 hours aftertransformation under conditions appropriate for the cell line to allowexpression of HLRRSI1. Phase microscopy is subsequently used to comparethe mitotic index of transformed versus control cells. An increase inthe mitotic index where HLRRSI1 stimulates cell proliferation indicatesapoptotic activity. Likewise, a decrease in cell numbers where HLRRSI1stimulates apoptosis indicates apoptotic activity.

[0810] The invention encompasses other assay methods known in the artand/or described herein.

Example 6 Method of Assessing the Ability of HLRRSI1 to ModulateCellular Adhesion

[0811] The role of the novel HLRRSI1 polypeptides in promoting celladhesion events could be determined by the generation of transfectedcell lines with the HLRRSI1 polynucleotides of the present invention,either transient or stable, and then subjected such cells to ahydrodynamic assay that can evaluate the relative importance of variousreceptor/ligand interactions in cell-cell and cell-substrate adhesion.Dynamic adhesion assays can simulate the forces found in the bloodstreamand may be used to estimate the strength of the bonds between cells andligands. Once representative assay is described by Jones et al, 1994.The skilled artisan would appreciate that this assay could readily beadapted to address the potential for HLRRSI1 to modulate cellularadhesion.

[0812] The invention encompasses other assay methods known in the artand/or described herein.

Example 7 Method of Assessing the Physiological Function of the HLRRSI1Polypeptide at the Cellular Level

[0813] The physiological function of the HLRRSI1 polypeptide may beassessed by expressing the sequences encoding HLRRSI1 at physiologicallyelevated levels in mammalian cell culture systems. cDNA is subclonedinto a mammalian expression vector containing a strong promoter thatdrives high levels of cDNA expression (examples are provided elsewhereherein). Vectors of choice include pCMV SPORT (Life Technologies) andpCR3.1 (Invitrogen, Carlsbad Calif.), both of which contain thecytomegalovirus promoter. 5-10, ug of recombinant vector are transientlytransfected into a human cell line, preferably of endothelial orhematopoietic origin, using either liposome formulations orelectroporation. 1-2 ug of an additional plasmid containing sequencesencoding a marker protein are cotransfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.Flow cytometry (FCM), an automated, laser optics-based technique, isused to identify transfected cells expressing GFP or CD64-GFP and toevaluate the apoptotic state of the cells and other cellular properties.FCM detects and quantifies the uptake of fluorescent molecules thatdiagnose events preceding or coincident with cell death. These eventsinclude changes in nuclear DNA content as measured by staining of DNAwith propidium iodide; changes in cell size and granularity as measuredby forward light scatter and 90 degree side light scatter;down-regulation of DNA synthesis as measured by decrease inbromodeoxyuridine uptake; alterations in expression of cell surface andintracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cvtometrv, Oxford, New York N.Y.

[0814] The influence of HLRRSI1 polypeptides on gene expression can beassessed using highly purified populations of cells transfected withsequences encoding HLRRSI1 and either CD64 or CD64-GFP. CD64 andCD64-GFP are expressed on the surface of transfected cells and bind toconserved regions of human immunoglobulin G (IgG). Transfected cells areefficiently separated from nontransfected cells using magnetic beadscoated with either human IgG or antibody against CD64 (DYNAL, LakeSuccess N.Y.). mRNA can be purified from the cells using methods wellknown by those of skill in the art. Expression of mRNA encoding HLRRSI1polypeptides and other genes of interest can be analyzed by northernanalysis or microarray techniques.

Example 8 Method of Screening for Compounds that Interact with theHLRRSI1 Polypeptide

[0815] The following assays are designed to identify compounds that bindto the HLRRSI1 polypeptide, bind to other cellular proteins thatinteract with the HLRRSI1 polypeptide, and to compounds that interferewith the interaction of the HLRRSI1 polypeptide with other cellularproteins.

[0816] Such compounds can include, but are not limited to, othercellular proteins. Specifically, such compounds can include, but are notlimited to, peptides, such as, for example, soluble peptides, including,but not limited to Ig-tailed fusion peptides, comprising extracellularportions of HLRRSI1 polypeptide transmembrane receptors, and members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghton, R. et al., 1991, Nature 354:84-86), made ofD-and/or L-configuration amino acids, phosphopeptides (including, butnot limited to, members of random or partially degenerate phosphopeptidelibraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′).sub.2 and FAb expression libary fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

[0817] Compounds identified via assays such as those described hereincan be useful, for example, in elaborating the biological function ofthe HLRRSI1 polypeptide, and for ameliorating symptoms of tumorprogression, for example. In instances, for example, whereby a tumorprogression state or disorder results from a lower overall level ofHLRRSI1 expression, HLRRSI1 polypeptide, and/or HLRRSI1 polypeptideactivity in a cell involved in the tumor progression state or disorder,compounds that interact with the HLRRSI1 polypeptide can include oneswhich accentuate or amplify the activity of the bound HLRRSI1polypeptide. Such compounds would bring about an effective increase inthe level of HLRRSI1 polypeptide activity, thus ameliorating symptoms ofthe tumor progression disorder or state. In instances whereby mutationswithin the HLRRSI1 polypeptide cause aberrant HLRRSI1 polypeptides to bemade which have a deleterious effect that leads to tumor progression,compounds that bind HLRRSI1 polypeptide can be identified that inhibitthe activity of the bound HLRRSI1 polypeptide. Assays for testing theeffectiveness of such compounds are known in the art and discussed,elsewhere herein.

Example 9 Method of Screening, In Vitro, Compounds that Bind to theHLRRSI1 Polypeptide

[0818] In vitro systems can be designed to identify compounds capable ofbinding the HLRRSI1 polypeptide of the invention. Compounds identifiedcan be useful, for example, in modulating the activity of wild typeand/or mutant HLRRSI1 polypeptide, preferably mutant HLRRSI1polypeptide, can be useful in elaborating the biological function of theHLRRSI1 polypeptide, can be utilized in screens for identifyingcompounds that disrupt normal HLRRSI1 polypeptide interactions, or canin themselves disrupt such interactions.

[0819] The principle of the assays used to identify compounds that bindto the HLRRSI1 polypeptide involves preparing a reaction mixture of theHLRRSI1 polypeptide and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringHLRRSI1 polypeptide or the test substance onto a solid phase anddetecting HLRRSI1 polypeptide/test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the HLRRSI1 polypeptide can be anchored onto a solid surface,and the test compound, which is not anchored, can be labeled, eitherdirectly or indirectly.

[0820] In practice, microtitre plates can conveniently be utilized asthe solid phase. The anchored component can be immobilized bynon-covalent or covalent attachments. Non-covalent attachment can beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized can beused to anchor the protein to the solid surface. The surfaces can beprepared in advance and stored.

[0821] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with alabeled anti-Ig antibody).

[0822] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for HLRRSI1polypeptide or the test compound to anchor any complexes formed insolution, and a labeled antibody specific for the other component of thepossible complex to detect anchored complexes.

Example 10 Method for Identifying a Putative Ligand for the HGCRBMY11Polypeptide

[0823] Ligand binding assays provide a direct method for ascertainingreceptor pharmacology and are adaptable to a high throughput format. Apanel of known leucine-rich repeat containing protein purified ligandsmay be radiolabeled to high specific activity (50-2000 Ci/mmol) forbinding studies. A determination is then made that the process ofradiolabeling does not diminish the activity of the ligand towards itsreceptor. Assay conditions for buffers, ions, pH and other modulatorssuch as nucleotides are optimized to establish a workable signal tonoise ratio for both membrane and whole cell receptor sources. For theseassays, specific receptor binding is defined as total associatedradioactivity minus the radioactivity measured in the presence of anexcess of unlabeled competing ligand. Where possible, more than onecompeting ligand is used to define residual nonspecific binding.

[0824] A number of leucine-rich repeat containing protein ligands areknown in the art and are encompassed by the present invention.

[0825] Alternatively, the HLRRSI1 polypeptide of the present inventionmay also be functionally screened against tissue extracts to identifynatural ligands. Extracts that produce positive functional responses canbe sequencially subfractionated until an activating ligand is isolatedidentified using methods well known in the art, some of which aredescribed herein.

Example 11 Method of Identifying Compounds that Interfere with HLRRSI1Polypeptide/Cellular Product Interaction

[0826] The HLRRSI1 polypeptide of the invention can, in vivo, interactwith one or more cellular or extracellular macromolecules, such asproteins. Such macromolecules include, but are not limited to,polypeptides, and those products identified via screening methodsdescribed, elsewhere herein. For the purposes of this discussion, suchcellular and extracellular macromolecules are referred to herein as“binding partner(s)”. For the purpose of the present invention, “bindingpartner” may also encompass polypeptides, small molecule compounds,polysaccarides, lipids, and any other molecule or molecule typereferenced herein. Compounds that disrupt such interactions can beuseful in regulating the activity of the HLRRSI1 polypeptide, especiallymutant HLRRSI1 polypeptide. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and the likedescribed in elsewhere herein.

[0827] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between the HLRRSI1polypeptide and its cellular or extracellular binding partner orpartners involves preparing a reaction mixture containing the HLRRSI1polypeptide, and the binding partner under conditions and for a timesufficient to allow the two products to interact and bind, thus forminga complex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of HLRRSI1polypeptide and its cellular or extracellular binding partner. Controlreaction mixtures are incubated without the test compound or with aplacebo. The formation of any complexes between the HLRRSI1 polypeptideand the cellular or extracellular binding partner is then detected. Theformation of a complex in the control reaction, but not in the reactionmixture containing the test compound, indicates that the compoundinterferes with the interaction of the HLRRSI1 polypeptide and theinteractive binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal HLRRSI1polypeptide can also be compared to complex formation within reactionmixtures containing the test compound and mutant HLRRSI1 polypeptide.This comparison can be important in those cases wherein it is desirableto identify compounds that disrupt interactions of mutant but not normalHLRRSI1 polypeptide.

[0828] The assay for compounds that interfere with the interaction ofthe HLRRSI1 polypeptide and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the HLRRSI1 polypeptide or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the HLRRSI1 polypeptide and the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with theHLRRSI1 polypeptide and interactive cellular or extracellular bindingpartner. Alternatively, test compounds that disrupt preformed complexes,e.g. compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

[0829] In a heterogeneous assay system, either the HLRRSI1 polypeptideor the interactive cellular or extracellular binding partner, isanchored onto a solid surface, while the non-anchored species islabeled, either directly or indirectly. In practice, microtitre platesare conveniently utilized. The anchored species can be immobilized bynon-covalent or covalent attachments. Non-covalent attachment can beaccomplished simply by coating the solid surface with a solution of theHLRRSI1 polypeptide or binding partner and drying. Alternatively, animmobilized antibody specific for the species to be anchored can be usedto anchor the species to the solid surface. The surfaces can be preparedin advance and stored.

[0830] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thenon-immobilized species is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe non-immobilized species is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the initially non-immobilized species (theantibody, in turn, can be directly labeled or indirectly labeled with alabeled anti-Ig antibody). Depending upon the order of addition ofreaction components, test compounds which inhibit complex formation orwhich disrupt preformed complexes can be detected.

[0831] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

[0832] In an alternate embodiment of the invention, a homogeneous assaycan be used. In this approach, a preformed complex of the HLRRSI1polypeptide and the interactive cellular or extracellular bindingpartner product is prepared in which either the HLRRSI1 polypeptide ortheir binding partners are labeled, but the signal generated by thelabel is quenched due to complex formation (see, e.g., U.S. Pat. No.4,109,496 by Rubenstein which utilizes this approach for immunoassays).

[0833] The addition of a test substance that competes with and displacesone of the species from the preformed complex will result in thegeneration of a signal above background. In this way, test substanceswhich disrupt HLRRSI1 polypeptide-cellular or extracellular bindingpartner interaction can be identified.

[0834] In a particular embodiment, the HLRRSI1 polypeptide can beprepared for immobilization using recombinant DNA techniques known inthe art. For example, the HLRRSI1 polypeptide coding region can be fusedto a glutathione-S-transferase (GST) gene using a fusion vector such aspGEX-5×-1, in such a manner that its binding activity is maintained inthe resulting fusion product. The interactive cellular or extracellularproduct can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above. Thisantibody can be labeled with the radioactive isotope .sup.125 I, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-HLRRSI1 polypeptide fusion product can be anchoredto glutathione-agarose beads. The interactive cellular or extracellularbinding partner product can then be added in the presence or absence ofthe test compound in a manner that allows interaction and binding tooccur. At the end of the reaction period, unbound material can be washedaway, and the labeled monoclonal antibody can be added to the system andallowed to bind to the complexed components. The interaction between theHLRRSI1 polypeptide and the interactive cellular or extracellularbinding partner can be detected by measuring the amount of radioactivitythat remains associated with the glutathione-agarose beads. A successfulinhibition of the interaction by the test compound will result in adecrease in measured radioactivity.

[0835] Alternatively, the GST-HLRRSI1 polypeptide fusion product and theinteractive cellular or extracellular binding partner product can bemixed together in liquid in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the bindingpartners are allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

[0836] In another embodiment of the invention, these same techniques canbe employed using peptide fragments that correspond to the bindingdomains of the HLRRSI1 polypeptide product and the interactive cellularor extracellular binding partner (in case where the binding partner is aproduct), in place of one or both of the full length products.

[0837] Any number of methods routinely practiced in the art can be usedto identify and isolate the protein's binding site. These methodsinclude, but are not limited to, mutagenesis of one of the genesencoding one of the products and screening for disruption of binding ina co-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can be selected. Sequenceanalysis of the genes encoding the respective products will reveal themutations that correspond to the region of the product involved ininteractive binding. Alternatively, one product can be anchored to asolid surface using methods described in this Section above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain can remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the cellular orextracellular binding partner product is obtained, short gene segmentscan be engineered to express peptide fragments of the product, which canthen be tested for binding activity and purified or synthesized.

Example 12 Isolation of a Specific Clone from the Deposited Sample

[0838] The deposited material in the sample assigned the ATCC DepositNumber cited in Table I for any given cDNA clone also may contain one ormore additional plasmids, each comprising a cDNA clone different fromthat given clone. Thus, deposits sharing the same ATCC Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table I.Typically, each ATCC deposit sample cited in Table I comprises a mixtureof approximately equal amounts (by weight) of about 1-10 plasmid DNAs,each containing a different cDNA clone and/or partial cDNA clone; butsuch a deposit sample may include plasmids for more or less than 2 cDNAclones.

[0839] Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table I.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:1.

[0840] Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-(-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

[0841] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:1 (i.e., within the region of SEQ ID NO:1 boundedby the 5′ NT and the 3′ NT of the clone defined in Table 1) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 ul of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at55 degree C. for 1 min; elongation at 72 degree C. for 1 min) areperformed with a Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the selected sequence by subcloning andsequencing the DNA product.

[0842] The polynucleotide(s) of the present invention, thepolynucleotide encoding the polypeptide of the present invention, or thepolypeptide encoded by the deposited clone may represent partial, orincomplete versions of the complete coding region (i.e., full-lengthgene). Several methods are known in the art for the identification ofthe 5′ or 3′ non-coding and/or coding portions of a gene which may notbe present in the deposited clone. The methods that follow are exemplaryand should not be construed as limiting the scope of the invention.These methods include but are not limited to, filter probing, cloneenrichment using specific probes, and protocols similar or identical to5′ and 3′ “RACE” protocols that are well known in the art. For instance,a method similar to 5′ RACE is available for generating the missing 5′end of a desired full-length transcript. (Fromont-Racine et al., NucleicAcids Res. 21(7):1683-1684 (1993)).

[0843] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

[0844] This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA that may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

[0845] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

[0846] An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988).Briefly, a cDNA clone missing either the 5′ or 3′ end can bereconstructed to include the absent base pairs extending to thetranslational start or stop codon, respectively. In some cases, cDNAsare missing the start of translation, therefor. The following brieflydescribes a modification of this original 5′ RACE procedure. Poly A+ ortotal RNAs reverse transcribed with Superscript II (Gibco/BRL) and anantisense or I complementary primer specific to the cDNA sequence. Theprimer is removed from the reaction with a Microcon Concentrator(Amicon). The first-strand cDNA is then tailed with dATP and terminaldeoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence isproduced which is needed for PCR amplification. The second strand issynthesized from the dA-tail in PCR buffer, Taq DNA polymerase(Perkin-Elmer Cetus), an oligo-dT primer containing three adjacentrestriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primercontaining just these restriction sites. This double-stranded cDNA isPCR amplified for 40 cycles with the same primers as well as a nestedcDNA-specific antisense primer. The PCR products are size-separated onan ethidium bromide-agarose gel and the region of gel containing cDNAproducts the predicted size of missing protein-coding DNA is removedcDNA is purified from the agarose with the Magic PCR Prep kit (Promega),restriction digested with XhoI or SalI, and ligated to a plasmid such aspBluescript SKII (Stratagene) at XhoI and EcORV sites. This DNA istransformed into bacteria and the plasmid clones sequenced to identifythe correct protein-coding inserts. Correct 5′ ends are confirmed bycomparing this sequence with the putatively identified homologue andoverlap with the partial cDNA clone. Similar methods known in the artand/or commercial kits are used to amplify and recover 3′ ends.

[0847] Several quality-controlled kits are commercially available forpurchase. Similar reagents and methods to those above are supplied inkit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of fulllength genes. A second kit is available from Clontech which is amodification of a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32(1991). The major differences in procedure are that the RNA isalkaline hydrolyzed after reverse transcription and RNA ligase is usedto join a restriction site-containing anchor primer to the first-strandcDNA. This obviates the necessity for the dA-tailing reaction whichresults in a polyT stretch that is difficult to sequence past.

[0848] An alternative to generating 5′ or 3′ cDNA from RNA is to usecDNA library double-stranded DNA. An asymmetric PCR-amplified antisensecDNA strand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

[0849] RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences toObtain Full Length Genes

[0850] Once a gene of interest is identified, several methods areavailable for the identification of the 5′ or 3′ portions of the genewhich may not be present in the original cDNA plasmid. These methodsinclude, but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar and identical to 5′ and 3′RACE.While the full-length gene may be present in the library and can beidentified by probing, a useful method for generating the 5′ or 3′ endis to use the existing sequence information from the original cDNA togenerate the missing information. A method similar to 5′RACE isavailable for generating the missing 5′ end of a desired full-lengthgene. (This method was published by Fromont-Racine et al., Nucleic AcidsRes., 21(7): 1683-1684 (1993)). Briefly, a specific RNA oligonucleotideis ligated to the 5′ ends of a population of RNA presumably 30containing full-length gene RNA transcript and a primer set containing aprimer specific to the ligated RNA oligonucleotide and a primer specificto a known sequence of the gene of interest, is used to PCR amplify the5′ portion of the desired full length gene which may then be sequencedand used to generate the full length gene. This method starts with totalRNA isolated from the desired source, poly A RNA may be used but is nota prerequisite for this procedure. The RNA preparation may then betreated with phosphatase if necessary to eliminate 5′ phosphate groupson degraded or damaged RNA which may interfere with the later RNA ligasestep. The phosphatase if used is then inactivated and the RNA is treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNApreparation can then be used as a template for first strand cDNAsynthesis using a gene specific oligonucleotide. The first strandsynthesis reaction can then be used as a template for PCR amplificationof the desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of theapoptosis related of interest. The resultant product is then sequencedand analyzed to confirm that the 5′ end sequence belongs to the relevantapoptosis related.

Example 13 Tissue Distribution of Polypeptide

[0851] Tissue distribution of mRNA expression of polynucleotides of thepresent invention is determined using protocols for Northern blotanalysis, described by, among others, Sambrook et al. For example, acDNA probe produced by the method described in Example 11 is labeledwith p32 using the rediprime TM DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using CHROMA SPINO-100 column (Clontech Laboratories,Inc.) according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various tissues for mRNAexpression.

[0852] Tissue Northern blots containing the bound mRNA of varioustissues are examined with the labeled probe using ExpressHybtmhybridization solution (Clonetech according to manufacturers protocolnumber PT1190-1. Northern blots can be produced using various protocolswell known in the art (e.g., Sambrook et al). Following hybridizationand washing, the blots are mounted and exposed to film at −70Covernight, and the films developed according to standard procedures.

Example 14 Chromosomal Mapping of the Polynucleotides

[0853] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:1. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions: 30 seconds, 95degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle isrepeated 32 times followed by one 5 minute cycle at 70 degree C.Mammalian DNA, preferably human DNA, is used as template in addition toa somatic cell hybrid panel containing individual chromosomes orchromosome fragments (Bios, Inc). The reactions are analyzed on either8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping isdetermined by the presence of an approximately 100 bp PCR fragment inthe particular somatic cell hybrid.

Example 15 Bacterial Expression of a Polypeptide

[0854] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 11, tosynthesize insertion fragments. The primers used to amplify the cDNAinsert should preferably contain restriction sites, such as BamHI andXbaI, at the 5′ end of the primers in order to clone the amplifiedproduct into the expression vector. For example, BamHI and XbaIcorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vectorencodes antibiotic resistance (Ampr), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

[0855] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, that expresses the lacIrepressor and also confers kanamycin resistance (Kanr). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[0856] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.600) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[0857] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4 degree C. The cell debris is removed bycentrifugation, and the supernatant containing the polypeptide is loadedonto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[0858] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[0859] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C. or frozen at −80 degree C.

Example 16 Purification of a Polypeptide from an Inclusion Body

[0860] The following alternative method can be used to purify apolypeptide expressed in E. coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 degree C. Upon completion of the production phaseof the E. coli fermentation, the cell culture is cooled to 4-10 degreeC. and the cells harvested by continuous centrifugation at 15,000 rpm(Heraeus Sepatech). On the basis of the expected yield of protein perunit weight of cell paste and the amount of purified protein required,an appropriate amount of cell paste, by weight, is suspended in a buffersolution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells aredispersed to a homogeneous suspension using a high shear mixer.

[0861] The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[0862] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

[0863] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4 degree C. without mixingfor 12 hours prior to further purification steps.

[0864] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 um membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[0865] Fractions containing the polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perceptive Biosystems) and weak anion (Poros CM-20, PerceptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A280 monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

[0866] The resultant polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Coomassie blue stained 16% SDS-PAGE gelwhen 5 ug of purified protein is loaded. The purified protein can alsobe tested for endotoxin/LPS contamination, and typically the LPS contentis less than 0.1 ng/ml according to LAL assays.

Example 17 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

[0867] In this example, the plasmid shuttle vector pAc373 is used toinsert a polynucleotide into a baculovirus to express a polypeptide. Atypical baculovirus expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV) followed by convenient restriction sites, which may include,for example BamHI, Xba I and Asp718. The polyadenylation site of thesimian virus 40 (“SV40”) is often used for efficient polyadenylation.For easy selection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedpolynucleotide.

[0868] Many other baculovirus vectors can be used in place of the vectorabove, such as pVL941 and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

[0869] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 11, tosynthesize insertion fragments. The primers used to amplify the cDNAinsert should preferably contain restriction sites at the 5′ end of theprimers in order to clone the amplified product into the expressionvector. Specifically, the cDNA sequence contained in the depositedclone, including the AUG initiation codon and the naturally associatedleader sequence identified elsewhere herein (if applicable), isamplified using the PCR protocol described in Example 11. If thenaturally occurring signal sequence is used to produce the protein, thevector used does not need a second signal peptide. Alternatively, thevector can be modified to include a baculovirus leader sequence, usingthe standard methods described in Summers et al., “A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555 (1987).

[0870] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[0871] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[0872] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[0873] Five ug of a plasmid containing the polynucleotide isco-transformed with 1.0 ug of a commercially available linearizedbaculovirus DNA (“BaculoGoldtm baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virusDNA and 5 ug of the plasmid are mixed in a sterile well of a microtiterplate containing 50 ul of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ulGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

[0874] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

[0875] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 uCi of ³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[0876] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the produced protein.

Example 18 Expression of a Polypeptide in Mammalian Cells

[0877] The polypeptide of the present invention can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

[0878] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0879] Alternatively, the polypeptide can be expressed in stable celllines containing the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

[0880] The transformed gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem . . . 253:1357-1370 (1978); Hamlin, J. L.and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J.and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992). Using these markers, the mammalian cells are grown inselective medium and the cells with the highest resistance are selected.These cell lines contain the amplified gene(s) integrated into achromosome. Chinese hamster ovary (CHO) and NSO cells are often used forthe production of proteins.

[0881] A polynucleotide of the present invention is amplified accordingto the protocol outlined in herein. If the naturally occurring signalsequence is used to produce the protein, the vector does not need asecond signal peptide. Alternatively, if the naturally occurring signalsequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891.) The amplifiedfragment is isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). Thefragment then is digested with appropriate restriction enzymes and againpurified on a 1% agarose gel.

[0882] The amplified fragment is then digested with the same restrictionenzyme and purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

[0883] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transformation. Five μg of an expression plasmid is cotransformedwith 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 19 Protein Fusions

[0884] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example described herein; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to the polypeptides of the presentinvention can target the protein to a specific subcellular localization,while covalent heterodimer or homodimers can increase or decrease theactivity of a fusion protein. Fusion proteins can also create chimericmolecules having more than one function. Finally, fusion proteins canincrease solubility and/or stability of the fused protein compared tothe non-fused protein. All of the types of fusion proteins describedabove can be made by modifying the following protocol, which outlinesthe fusion of a polypeptide to an IgG molecule.

[0885] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector. Note that the polynucleotideis cloned without a stop codon, otherwise a fusion protein will not beproduced.

[0886] The naturally occurring signal sequence may be used to producethe protein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.) Human IgG Fc region:GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACA (SEQ ID NO:28)CATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGC GACTCTAGAGGAT

Example 20 Production of an Antibody from a Polypeptide

[0887] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing a polypeptide of the present inventionare administered to an animal to induce the production of seracontaining polyclonal antibodies. In a preferred method, a preparationof the protein is prepared and purified to render it substantially freeof natural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

[0888] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur.J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involveimmunizing an animal (preferably a mouse) with polypeptide or, morepreferably, with a polypeptide-expressing cell. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56degrees C.), and supplemented with about 10 g/l of nonessential aminoacids, about 1,000 U/ml of penicillin, and about 100 ug/ml ofstreptomycin.

[0889] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981).) Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding thepolypeptide.

[0890] Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodythat binds to a second antibody. In accordance with this method, proteinspecific antibodies are used to immunize an animal, preferably a mouse.The splenocytes of such an animal are then used to produce hybridomacells, and the hybridoma cells are screened to identify clones thatproduce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

[0891] It will be appreciated that Fab and F(ab′)₂ and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). Alternatively,protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

[0892] For in vivo use of antibodies in humans, it may be preferable touse “humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).) Moreover, in another preferred method, theantibodies directed against the polypeptides of the present inventionmay be produced in plants. Specific methods are disclosed in U.S. Pat.Nos. 5,959,177, and 6,080,560, which are hereby incorporated in theirentirety herein. The methods not only describe methods of expressingantibodies, but also the means of assembling foreign multimeric proteinsin plants (i.e., antibodies, etc,), and the subsequent secretion of suchantibodies from the plant.

Example 21 Regulation of Protein Expression Via Controlled Aggregationin the Endoplasmic Reticulum

[0893] As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

[0894] Numerous methods have been developed to date for introducingforeign genes, either under the control of an inducible, constitutivelyactive, or endogenous promoter, into organisms. Of particular interestare the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad.Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc,.), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

[0895] A new alternative method of controlling gene expression levels ofa protein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830, (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

[0896] Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

[0897] Fusion protein constructs are created using polynucleotidesequences of the present invention with one or more copies (preferablyat least 2, 3, 4, or more) of a conditional aggregation domain (CAD) adomain that interacts with itself in a ligand-reversible manner (i.e.,in the presence of an inducing agent) using molecular biology methodsknown in the art and discussed elsewhere herein. The CAD domain may bethe mutant domain isolated from the human FKBP12 (Phe³⁶ to Met) protein(as disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)_(x) domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

[0898] The specific methods described herein for controlling proteinsecretion levels through controlled ER aggregation are not meant to belimiting are would be generally applicable to any of the polynucleotidesand polypeptides of the present invention, including variants,homologues, orthologs, and fragments therein.

Example 22 Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

[0899] Many eukaryotic cell surface and proteins arepost-translationally processed to incorporate N-linked and O-linkedcarbohydrates (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem.54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838).Protein glycosylation is thought to serve a variety of functionsincluding: augmentation of protein folding, inhibition of proteinaggregation, regulation of intracellular trafficking to organelles,increasing resistance to proteolysis, modulation of proteinantigenicity, and mediation of intercellular adhesion (Fieldler andSimons (1995) Cell, 81:309-312; Helenius (1994) Mol. Biol. Of the Cell5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982)Cell, 37:417-427; Alexamnder and Elder (1984), Science, 226:1328-1330;and Flack et al., (1994), J. Biol. Chem . . . , 269:14015-14020). Inhigher organisms, the nature and extent of glycosylation can markedlyaffect the circulating half-life and bio-availability of proteins bymechanisms involving receptor mediated uptake and clearance (Ashwell andMorrell, (1974), Adv. Enzymol., 41:99-128; Ashwell and Harford (1982),Ann. Rev. Biochem., 51:531-54). Receptor systems have been identifiedthat are thought to play a major role in the clearance of serum proteinsthrough recognition of various carbohydrate structures on theglycoproteins (Stockert (1995), Physiol. Rev., 75:591-609; Kery et al.,(1992), Arch. Biochem. Biophys., 298:49-55). Thus, production strategiesresulting in incomplete attachment of terminal sialic acid residuesmight provide a means of shortening the bioavailability and half-life ofglycoproteins. Conversely, expression strategies resulting in saturationof terminal sialic acid attachment sites might lengthen proteinbioavailability and half-life.

[0900] In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol. Chem., 263:5955-5960; Gallagher et al.,(1992), J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem . . . 263:17516-17521).While glycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

[0901] Moreover, it is unclear to what extent a glycosylation site inone species will be recognized by another species glycosylationmachinery. Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E. coli, yeast, orviral organisms; or an E. coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

[0902] A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

[0903] The skilled artisan would acknowledge the existence of othercomputer algorithms capable of predicting the location of glycosylationsites within a protein. For example, the Motif computer program(Genetics Computer Group suite of programs) provides this function, aswell.

Example 23 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

[0904] Although many of the most biologically active proteins known arehighly effective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

[0905] Thus, one aspect of the present invention relates to the abilityto enhance specific characteristics of invention through directedmolecular evolution. Such an enhancement may, in a non-limiting example,benefit the inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

[0906] For example, an engineered leucine-rich repeat protein may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered leucine-rich repeat protein may be constitutively activein the absence of ligand binding. In yet another example, an engineeredleucine-rich repeat protein may be capable of being activated with lessthan all of the regulatory factors and/or conditions typically requiredfor leucine-rich repeat protein activation (e.g., ligand binding,phosphorylation, conformational changes, etc.). Such leucine-rich repeatproteins would be useful in screens to identify leucine-rich repeatprotein modulators, among other uses described herein.

[0907] Directed evolution is comprised of several steps. The first stepis to establish a library of variants for the gene or protein ofinterest. The most important step is to then select for those variantsthat entail the activity you wish to identify. The design of the screenis essential since your screen should be selective enough to eliminatenon-useful variants, but not so stringent as to eliminate all variants.The last step is then to repeat the above steps using the best variantfrom the previous screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

[0908] Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

[0909] Random mutagenesis has been the most widely recognized method todate. Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

[0910] While both of the aforementioned methods are effective forcreating randomized pools of macromolecule variants, a third method,termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747,(1994)) has recently been elucidated. DNA shuffling has also beenreferred to as “directed molecular evolution”, “exon-shuffling”,“directed enzyme evolution”, “in vitro evolution”, and “artificialevolution”. Such reference terms are known in the art and areencompassed by the invention. This new, preferred, method apparentlyovercomes the limitations of the previous methods in that it not onlypropagates positive traits, but simultaneously eliminates negativetraits in the resulting progeny.

[0911] DNA shuffling accomplishes this task by combining the principalof in vitro recombination, along with the method of “error-prone” PCR.In effect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments —further diversifying the potential hybridization sitesduring the annealing step of the reaction.

[0912] A variety of reaction conditions could be utilized to carry-outthe DNA shuffling reaction. However, specific reaction conditions forDNA shuffling are provided, for example, in PNAS, 91:10747, (1994).Briefly:

[0913] Prepare the DNA substrate to be subjected to the DNA shufflingreaction. Preparation may be in the form of simply purifying the DNAfrom contaminating cellular material, chemicals, buffers,oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entailthe use of DNA purification kits as those provided by Qiagen, Inc., orby the Promega, Corp., for example.

[0914] Once the DNA substrate has been purified, it would be subjectedto Dnase I digestion. About 2-4 ug of the DNA substrate(s) would bedigested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mMTris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. Theresulting fragments of 10-50 bp could then be purified by running themthrough a 2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

[0915] The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30s, 50 C for 30s, and72 C for 30s). The referred primers would be primers corresponding tothe nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

[0916] The resulting shuffled, assembled, and amplified product can bepurified using methods well known in the art (e.g., Qiagen PCRpurification kits) and then subsequently cloned using appropriaterestriction enzymes.

[0917] Although a number of variations of DNA shuffling have beenpublished to date, such variations would be obvious to the skilledartisan and are encompassed by the invention. The DNA shuffling methodcan also be tailored to the desired level of mutagenesis using themethods described by Zhao, et al. (Nucl Acid Res., 25(6):1307-1308,(1997).

[0918] As described above, once the randomized pool has been created, itcan then be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

[0919] DNA shuffling has several advantages. First, it makes use ofbeneficial mutations. When combined with screening, DNA shuffling allowsthe discovery of the best mutational combinations and does not assumethat the best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

[0920] A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

[0921] Finally, recombination enables parallel processing. Thisrepresents a significant advantage since there are likely multiplecharacteristics that would make a protein more desirable (e.g.solubility, activity, etc.). Since it is increasingly difficult toscreen for more than one desirable trait at a time, other methods ofmolecular evolution tend to be inhibitory. However, using recombination,it would be possible to combine the randomized fragments of the bestrepresentative variants for the various traits, and then select formultiple properties at once.

[0922] DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

[0923] Likewise, the invention encompasses the application of DNAshuffling technology to the evolution of polynucleotides andpolypeptides of the invention, wherein one or more cycles of DNAshuffling include, in addition to the gene template DNA,oligonucleotides coding for known allelic sequences, optimized codonsequences, known variant sequences, known polynucleotide polymorphismsequences, known ortholog sequences, known homologue sequences,additional homologous sequences, additional non-homologous sequences,sequences from another species, and any number and combination of theabove.

[0924] In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

[0925] Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 24 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

[0926] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:1. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70degrees C., using buffer solutions described in Sidransky et al.,Science 252:706 (1991).

[0927] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

[0928] PCR products is cloned into T-tailed vectors as described inHolton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced withT7 polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

[0929] Genomic rearrangements are also observed as a method ofdetermining alterations in a gene corresponding to a polynucleotide.Genomic clones isolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

[0930] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 25 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

[0931] A polypeptide of the present invention can be detected in abiological sample, and if an increased or decreased level of thepolypeptide is detected, this polypeptide is a marker for a particularphenotype. Methods of detection are numerous, and thus, it is understoodthat one skilled in the art can modify the following assay to fit theirparticular needs.

[0932] For example, antibody-sandwich ELISAs are used to detectpolypeptides in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced by the method described elsewhere herein.The wells are blocked so that non-specific binding of the polypeptide tothe well is reduced.

[0933] The coated wells are then incubated for >2 hours at RT with asample containing the polypeptide. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedpolypeptide.

[0934] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[0935] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot polypeptide concentration on theX-axis (log scale) and fluorescence or absorbance of the Y-axis (linearscale). Interpolate the concentration of the polypeptide in the sampleusing the standard curve.

Example 26 Formulation

[0936] The invention also provides methods of treatment and/orprevention diseases, disorders, and/or conditions (such as, for example,any one or more of the diseases or disorders disclosed herein) byadministration to a subject of an effective amount of a Therapeutic. Bytherapeutic is meant a polynucleotides or polypeptides of the invention(including fragments and variants), agonists or antagonists thereof,and/or antibodies thereto, in combination with a pharmaceuticallyacceptable carrier type (e.g., a sterile carrier).

[0937] The Therapeutic will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the Therapeutic alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[0938] As a general proposition, the total pharmaceutically effectiveamount of the Therapeutic administered parenterally per dose will be inthe range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Therapeutic is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0939] Therapeutics can be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

[0940] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

[0941] Therapeutics of the invention may also be suitably administeredby sustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[0942] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[0943] Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see, generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

[0944] In yet an additional embodiment, the Therapeutics of theinvention are delivered by way of a pump (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[0945] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0946] For parenteral administration, in one embodiment, the Therapeuticis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

[0947] Generally, the formulations are prepared by contacting theTherapeutic uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0948] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0949] The Therapeutic will typically be formulated in such vehicles ata concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml,at a pH of about 3 to 8. It will be understood that the use of certainof the foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[0950] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

[0951] Therapeutics ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

[0952] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the Therapeutics of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theTherapeutics may be employed in conjunction with other therapeuticcompounds.

[0953] The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0954] The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0955] In one embodiment, the Therapeutics of the invention areadministered in combination with members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with theTherapeutics of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[0956] In certain embodiments, Therapeutics of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to,RETROVIR((zidovudine/AZT), VIDEX((didanosine/ddI), HIVID((zalcitabine/ddC), ZERIT((stavudine/d4T), EPIVIR((lamivudine/3TC), andCOMBIVIR( (zidovudine/lamivudine). Non-nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to,VIRAMUNE((nevirapine), RESCRIPTOR((delavirdine), andSUSTIVA((efavirenz). Protease inhibitors that may be administered incombination with the Therapeutics of the invention, include, but are notlimited to, CRIXIVAN((indinavir), NORVIR((ritonavir),INVIRASE((saquinavir), and VIRACEPT((nelfinavir). In a specificembodiment, antiretroviral agents, nucleoside reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors, and/orprotease inhibitors may be used in any combination with Therapeutics ofthe invention to treat AIDS and/or to prevent or treat HIV infection.

[0957] In other embodiments, Therapeutics of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, ATOVAQUONE(,ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, ETHAMBUTOL(, RIFABUTIN(,CLARITHROMYCIN(, AZITHROMYCIN(, GANCICLOVIR(, FOSCARNET(, CIDOFOVIR(,FLUCONAZOLE(, ITRACONAZOLE(, KETOCONAZOLE(, ACYCLOVIR(, FAMCICOLVIR(,PYRIMETHAMINE(, LEUCOVORIN(, NEUPOGEN( (filgrastim/G-CSF), andLEUKINE((sargramostim/GM-CSF). In a specific embodiment, Therapeutics ofthe invention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, and/orATOVAQUONE(to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, and/or ETHAMBUTOL(toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN(, CLARITHROMYCIN(,and/or AZITHROMYCIN(to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR(, FOSCARNET(, and/or CIDOFOVIR(to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE(, ITRACONAZOLE(, and/or KETOCONAZOLE(toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR(and/or FAMCICOLVIR(to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE(and/orLEUCOVORIN(to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN(and/or NEUPOGEN(to prophylactically treat or prevent anopportunistic bacterial infection.

[0958] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[0959] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

[0960] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0961] In specific embodiments, Therapeutics of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the Therapeutics of theinvention include, but are not limited to, ORTHOCLONE((OKT3),SANDIMMUNE(/NEORAL(/SANGDYA((cyclosporin), PROGRAF((tacrolimus),CELLCEPT((mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE( (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

[0962] In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR(, IVEEGAM(, SANDOGLOBULIN(, GAMMAGARD S/D(, andGAMIMUNE(. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

[0963] In an additional embodiment, the Therapeutics of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with theTherapeutics of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0964] In another embodiment, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0965] In a specific embodiment, Therapeutics of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, Therapeutics of the invention areadministered in combination with Rituximab. In a further embodiment,Therapeutics of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[0966] In an additional embodiment, the Therapeutics of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

[0967] In an additional embodiment, the Therapeutics of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[0968] In an additional embodiment, the Therapeutics of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE((SARGRAMOSTIM( ) and NEUPOGEN((FILGRASTIM( ).

[0969] In an additional embodiment, the Therapeutics of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the Therapeuticsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0970] In a specific embodiment, formulations of the present inventionmay further comprise antagonists of P-glycoprotein (also referred to asthe multiresistance protein, or PGP), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). P-glycoprotein is well known for decreasingthe efficacy of various drug administrations due to its ability toexport intracellular levels of absorbed drug to the cell exterior. Whilethis activity has been particularly pronounced in cancer cells inresponse to the administration of chemotherapy regimens, a variety ofother cell types and the administration of other drug classes have beennoted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations inthe PGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each parent—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

[0971] Moreover, in another specific embodiment, formulations of thepresent invention may further comprise antagonists of OATP2 (alsoreferred to as the multiresistance protein, or MRP2), includingantagonists of its encoding polynucleotides (e.g., antisenseoligonucleotides, ribozymes, zinc-finger proteins, etc.). The inventionalso further comprises any additional antagonists known to inhibitproteins thought to be attributable to a multidrug resistant phenotypein proliferating cells.

[0972] In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 27 Method of Treating Decreased Levels of the Polypeptide

[0973] The present invention relates to a method for treating anindividual in need of an increased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an agonistof the invention (including polypeptides of the invention). Moreover, itwill be appreciated that conditions caused by a decrease in the standardor normal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

[0974] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided herein.

Example 28 Method of Treating Increased Levels of the Polypeptide

[0975] The present invention also relates to a method of treating anindividual in need of a decreased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of anantagonist of the invention (including polypeptides and antibodies ofthe invention).

[0976] In one example, antisense technology is used to inhibitproduction of a polypeptide of the present invention. This technology isone example of a method of decreasing levels of a polypeptide,preferably a secreted form, due to a variety of etiologies, such ascancer. For example, a patient diagnosed with abnormally increasedlevels of a polypeptide is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the treatmentwas well tolerated. The formulation of the antisense polynucleotide isprovided herein.

Example 29 Method of Treatment Using Gene Therapy—Ex Vivo

[0977] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degreeC. for approximately one week.

[0978] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0979] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcORI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0980] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 11 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcORI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcORI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[0981] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0982] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[0983] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 30 Gene Therapy Using Endogenous Genes Corresponding toPolynucleotides of the Invention

[0984] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

[0985] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

[0986] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0987] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0988] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous polynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

[0989] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

[0990] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the locus correspondingto the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas,Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplifiedby PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end.Two non-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

[0991] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0992] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[0993] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 31 Method of Treatment Using Gene Therapy—In Vivo

[0994] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques and methods areknown in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

[0995] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0996] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

[0997] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[0998] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[0999] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[1000] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[1001] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[1002] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 32 Transgenic Animals

[1003] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[1004] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[1005] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[1006] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[1007] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[1008] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[1009] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of polypeptides of the present invention, studyingdiseases, disorders, and/or conditions associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch diseases, disorders, and/or conditions.

Example 33 Knock-Out Animals

[1010] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

[1011] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[1012] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[1013] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[1014] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 34 Production of an Antibody

[1015] a) Hybridoma Technology

[1016] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing HLRRSI1 are administered to an animalto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of HLRRSI1 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

[1017] Monoclonal antibodies specific for protein HLRRSI1 are preparedusing hybridoma technology. (Kohler et al., Nature 256:495 (1975);Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, ananimal (preferably a mouse) is immunized with HLRRSI1 polypeptide or,more preferably, with a secreted HLRRSI1 polypeptide-expressing cell.Such polypeptide-expressing cells are cultured in any suitable tissueculture medium, preferably in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

[1018] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the HLRRSI1polypeptide.

[1019] Alternatively, additional antibodies capable of binding toHLRRSI1 polypeptide can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody that binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theHLRRSI1 protein-specific antibody can be blocked by HLRRSI1. Suchantibodies comprise anti-idiotypic antibodies to the HLRRSI1protein-specific antibody and are used to immunize an animal to induceformation of further HLRRSI1 protein-specific antibodies.

[1020] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed herein. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).)

[1021] b) Isolation of Antibody Fragments Directed

[1022] Against HLRRSI1 from a Library of scFvs

[1023] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against HLRRSI1 to which the donor may or may not have beenexposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

[1024] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in PCT publication WO 92/01047. To rescuephage displaying antibody fragments, approximately 109 E. coli harboringthe phagemid are used to inoculate 50 ml of 2×TY containing 1% glucoseand 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8with shaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

[1025] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[1026] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[1027] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Example 35 Assays Detecting Stimulation or Inhibition of B CellProliferation and Differentiation

[1028] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,1L-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

[1029] One of the best studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

[1030] In Vitro Assay—Purified polypeptides of the invention, ortruncated forms thereof, is assessed for its ability to induceactivation, proliferation, differentiation or inhibition and/or death inB-cell populations and their precursors. The activity of thepolypeptides of the invention on purified human tonsillar B cells,measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, isassessed in a standard B-lymphocyte co-stimulation assay in whichpurified tonsillar B cells are cultured in the presence of eitherformalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilizedanti-human IgM antibody as the priming agent. Second signals such asIL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cellproliferation as measured by tritiated-thymidine incorporation. Novelsynergizing agents can be readily identified using this assay. The assayinvolves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).

[1031] Various dilutions of each sample are placed into individual wellsof a 96-well plate to which are added 105 B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U/ml penicillin,10 ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[1032] In Vivo Assay—BALB/c mice are injected (i.p.) twice per day withbuffer only, or 2 mg/Kg of a polypeptide of the invention, or truncatedforms thereof. Mice receive this treatment for 4 consecutive days, atwhich time they are sacrificed and various tissues and serum collectedfor analyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

[1033] Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

[1034] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

[1035] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 36 T Cell Proliferation Assay

[1036] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of 3H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 (l/well of mAb toCD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnightat 4 degrees C. (1 (g/ml in 0.05M bicarbonate buffer, pH 9.5), thenwashed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×104/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of polypeptides of theinvention (total volume 200 ul). Relevant protein buffer and mediumalone are controls. After 48 hr. culture at 37 degrees C., plates arespun for 2 min. at 1000 rpm and 100 (1 of supernatant is removed andstored −20 degrees C. for measurement of IL-2 (or other cytokines) ifeffect on proliferation is observed. Wells are supplemented with 100 ulof medium containing 0.5 uCi of 3H-thymidine and cultured at 37 degreesC. for 18-24 hr. Wells are harvested and incorporation of 3H-thymidineused as a measure of proliferation. Anti-CD3 alone is the positivecontrol for proliferation. IL-2 (100 U/ml) is also used as a controlwhich enhances proliferation. Control antibody which does not induceproliferation of T cells is used as the negative controls for theeffects of polypeptides of the invention.

[1037] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 37 Effect of Polypeptides of the Invention on the Expression ofMHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

[1038] Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-(,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFC(RII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

[1039] FACS analysis of surface antigens is performed as follows. Cellsare treated 1-3 days with increasing concentrations of polypeptides ofthe invention or LPS (positive control), washed with PBS containing 1%BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

[1040] Effect on the production of cytokines. Cytokines generated bydendritic cells, in particular IL-12, are important in the initiation ofT-cell dependent immune responses. IL-12 strongly influences thedevelopment of Th1 helper T-cell immune response, and induces cytotoxicT and NK cell function. An ELISA is used to measure the IL-12 release asfollows. Dendritic cells (106/ml) are treated with increasingconcentrations of polypeptides of the invention for 24 hours. LPS (100ng/ml) is added to the cell culture as positive control. Supernatantsfrom the cell cultures are then collected and analyzed for IL-12 contentusing commercial ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)).The standard protocols provided with the kits are used.

[1041] Effect on the expression of MHC Class II, costimulatory andadhesion molecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

[1042] FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

[1043] Monocyte activation and/or increased survival. Assays formolecules that activate (or alternatively, inactivate) monocytes and/orincrease monocyte survival (or alternatively, decrease monocytesurvival) are known in the art and may routinely be applied to determinewhether a molecule of the invention functions as an inhibitor oractivator of monocytes. Polypeptides, agonists, or antagonists of theinvention can be screened using the three assays described below. Foreach of these assays, Peripheral blood mononuclear cells (PBMC) arepurified from single donor leukopacks (American Red Cross, Baltimore,Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytesare isolated from PBMC by counterflow centrifugal elutriation.

[1044] Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the compound to be tested. Cells aresuspended at a concentration of 2×106/ml in PBS containing PI at a finalconcentration of 5 (g/ml, and then incubated at room temperature for 5minutes before FACScan analysis. PI uptake has been demonstrated tocorrelate with DNA fragmentation in this experimental paradigm.

[1045] Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×105 cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/ml) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24 h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit(e.g., R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

[1046] Oxidative burst. Purified monocytes are plated in 96-w plate at2-1×105 cell/well. Increasing concentrations of polypeptides of theinvention are added to the wells in a total volume of 0.2 ml culturemedium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 daysincubation, the plates are centrifuged and the medium is removed fromthe wells. To the macrophage monolayers, 0.2 ml per well of phenol redsolution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mMdextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMA). The plates are incubated at 37(C for 2hours and the reaction is stopped by adding 20 μl 1N NaOH per well. Theabsorbance is read at 610 nm. To calculate the amount of H₂O₂ producedby the macrophages, a standard curve of a H₂O₂ solution of knownmolarity is performed for each experiment.

[1047] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 38 Biological Effects of HLRRSI1 Polypeptides of the Invention

[1048] Astrocyte and Neuronal Assays.

[1049] Recombinant polypeptides of the invention, expressed inEscherichia coli and purified as described above, can be tested foractivity in promoting the survival, neurite outgrowth, or phenotypicdifferentiation of cortical neuronal cells and for inducing theproliferation of glial fibrillary acidic protein immunopositive cells,astrocytes. The selection of cortical cells for the bioassay is based onthe prevalent expression of FGF-1 and FGF-2 in cortical structures andon the previously reported enhancement of cortical neuronal survivalresulting from FGF-2 treatment. A thymidine incorporation assay, forexample, can be used to elucidate a polypeptide of the invention'sactivity on these cells.

[1050] Moreover, previous reports describing the biological effects ofFGF-2 (basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

[1051] Fibroblast and Endothelial Cell Assays.

[1052] Human lung fibroblasts are obtained from Clonetics (San Diego,Calif.) and maintained in growth media from Clonetics. Dermalmicrovascular endothelial cells are obtained from Cell Applications (SanDiego, Calif.). For proliferation assays, the human lung fibroblasts anddermal microvascular endothelial cells can be cultured at 5,000cells/well in a 96-well plate for one day in growth medium. The cellsare then incubated for one day in 0.1% BSA basal medium. After replacingthe medium with fresh 0.1% BSA medium, the cells are incubated with thetest proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) is added to each well to a final concentration of 10%. The cellsare incubated for 4 hr. Cell viability is measured by reading in aCytoFluor fluorescence reader. For the PGE2 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or polypeptides of the invention with or withoutIL-1 (for 24 hours. The supernatants are collected and assayed for PGE2by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the humanlung fibroblasts are cultured at 5,000 cells/well in a 96-well plate forone day. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1 (for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

[1053] Human lung fibroblasts are cultured with FGF-2 or polypeptides ofthe invention for 3 days in basal medium before the addition of AlamarBlue to assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

[1054] Parkinson Models.

[1055] The loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP+is actively accumulated in dopaminergic neurons by thehigh-affinity reuptake transporter for dopamine. MPP+is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

[1056] It has been demonstrated in tissue culture paradigms that FGF-2(basic FGF) has trophic activity towards nigral dopaminergic neurons(Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

[1057] Based on the data with FGF-2, polypeptides of the invention canbe evaluated to determine whether it has an action similar to that ofFGF-2 in enhancing dopaminergic neuronal survival in vitro and it canalso be tested in vivo for protection of dopaminergic neurons in thestriatum from the damage associated with MPTP treatment. The potentialeffect of a polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopaminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

[1058] Since the dopaminergic neurons are isolated from animals atgestation day 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

[1059] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 39 The Effect of the HLRRSI1 Polypeptides of the Invention onthe Growth of Vascular Endothelial Cells

[1060] On day 1, human umbilical vein endothelial cells (HUVEC) areseeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/mlendothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day2, the medium is replaced with M199 containing 10% FBS, 8 units/mlheparin. A polypeptide having the amino acid sequence of SEQ ID NO:2,and positive controls, such as VEGF and basic FGF (bFGF) are added, atvarying concentrations. On days 4 and 6, the medium is replaced. On day8, cell number is determined with a Coulter Counter.

[1061] An increase in the number of HUVEC cells indicates that thepolypeptide of the invention may proliferate vascular endothelial cells.

[1062] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 40 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

[1063] For evaluation of mitogenic activity of growth factors, thecolorimetric MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

[1064] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 41 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

[1065] HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposedto denaturing solution and then incubated with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem . . .6:271(36):21985-21992 (1996).

[1066] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 42 Stimulation of Endothelial Migration

[1067] This example will be used to explore the possibility that apolypeptide of the invention may stimulate lymphatic endothelial cellmigration.

[1068] Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO₂ to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, IL). Migration is quantifiedby counting cells of three random high-power fields (40×) in each well,and all groups are performed in quadruplicate.

[1069] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 43 Stimulation of Nitric Oxide Production by Endothelial Cells

[1070] Nitric oxide released by the vascular endothelium is believed tobe a mediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

[1071] Nitric oxide is measured in 96-well plates of confluentmicrovascular endothelial cells after 24 hours starvation and asubsequent 4 hr exposure to various levels of a positive control (suchas VEGF-1) and the polypeptide of the invention. Nitric oxide in themedium is determined by use of the Griess reagent to measure totalnitrite after reduction of nitric oxide-derived nitrate by nitratereductase. The effect of the polypeptide of the invention on nitricoxide release is examined on HUVEC.

[1072] Briefly, NO release from cultured HUVEC monolayer is measuredwith a NO-specific polarographic electrode connected to a NO meter(Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NOelements is performed according to the following equation:

2 KNO2+2 KI+2H2SO4 6 2 NO+I2+2H20+2 K2SO4

[1073] The standard calibration curve is obtained by adding gradedconcentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing K1 and H2SO4. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×106 endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

[1074] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 44 Effect of HLRRSI1 Polypepides of the Invention on CordFormation in Angiogenesis

[1075] Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

[1076] CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications'CADMEC Growth Medium and used at passage 5. For thein vitro angiogenesis assay, the wells of a 48-well cell culture plateare coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications'Chord FormationMedium containing control buffer or a polypeptide of the invention (0.1to 100 ng/ml) and the cells are cultured for an additional 48 hr. Thenumbers and lengths of the capillary-like chords are quantitated throughuse of the Boeckeler VIA-170 video image analyzer. All assays are donein triplicate.

[1077] Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

[1078] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 45 Angiogenic Effect on Chick Chorioallantoic Membrane

[1079] Chick chorioallantoic membrane (CAM) is a well-established systemto examine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of polypeptides of the invention tostimulate angiogenesis in CAM can be examined.

[1080] Fertilized eggs of the White Leghorn chick (Gallus gallus) andthe Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

[1081] On Day 4 of development, a window is made into the egg shell ofchick eggs. The embryos are checked for normal development and the eggssealed with cellotape. They are further incubated until Day 13.Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks ofabout 5 mm in diameter. Sterile and salt-free growth factors aredissolved in distilled water and about 3.3 mg/5 ml are pipetted on thedisks. After air-drying, the inverted disks are applied on CAM. After 3days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehydeand rinsed in 0.12 M sodium cacodylate buffer. They are photographedwith a stereo microscope [Wild M8] and embedded for semi- and ultrathinsectioning as described above. Controls are performed with carrier disksalone.

[1082] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 46 Angiogenesis Assay Using a Matrigel Implant in Mouse

[1083] In vivo angiogenesis assay of a polypeptide of the inventionmeasures the ability of an existing capillary network to form newvessels in an implanted capsule of murine extracellular matrix material(Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C.and the mixture is then injected subcutaneously in mice where itsolidifies. After 7 days, the solid “plug” of Matrigel is removed andexamined for the presence of new blood vessels. Matrigel is purchasedfrom Becton Dickinson Labware/Collaborative Biomedical Products.

[1084] When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 miceapproximately 8 weeks old are injected with the mixture of Matrigel andexperimental protein at 2 sites at the midventral aspect of the abdomen(0.5 ml/site). After 7 days, the mice are sacrificed by cervicaldislocation, the Matrigel plugs are removed and cleaned (i.e., allclinging membranes and fibrous tissue is removed). Replicate whole plugsare fixed in neutral buffered 10% formaldehyde, embedded in paraffin andused to produce sections for histological examination after stainingwith Masson's Trichrome. Cross sections from 3 different regions of eachplug are processed. Selected sections are stained for the presence ofvWF. The positive control for this assay is bovine basic FGF (150ng/ml). Matrigel alone is used to determine basal levels ofangiogenesis.

[1085] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 47 Rescue of Ischemia in Rabbit Lower Limb Model

[1086] To study the in vivo effects of polynucleotides and polypeptidesof the invention on ischemia, a rabbit hindlimb ischemia model iscreated by surgical removal of one femoral arteries as describedpreviously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). Theexcision of the femoral artery results in retrograde propagation ofthrombus and occlusion of the external iliac artery. Consequently, bloodflow to the ischemic limb is dependent upon collateral vesselsoriginating from the internal iliac artery (Takeshitaet al. Am J. Pathol147:1649-1660 (1995)). An interval of 10 days is allowed forpost-operative recovery of rabbits and development of endogenouscollateral vessels. At 10 day post-operatively (day 0), after performinga baseline angiogram, the internal iliac artery of the ischemic limb istransfected with 500 mg naked expression plasmid containing apolynucleotide of the invention by arterial gene transfer technologyusing a hydrogel-coated balloon catheter as described (Riessen et al.Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90:936-944 (1992)). When a polypeptide of the invention is used in thetreatment, a single bolus of 500 mg polypeptide of the invention orcontrol is delivered into the internal iliac artery of the ischemic limbover a period of 1 min. through an infusion catheter. On day 30, variousparameters are measured in these rabbits: (a) BP ratio—The bloodpressure ratio of systolic pressure of the ischemic limb to that ofnormal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flowduring undilated condition and Max FL: the blood flow during fullydilated condition (also an indirect measure of the blood vessel amount)and Flow Reserve is reflected by the ratio of max FL: resting FL; (c)Angiographic Score—This is measured by the angiogram of collateralvessels. A score is determined by the percentage of circles in anoverlaying grid that with crossing opacified arteries divided by thetotal number m the rabbit thigh; (d) Capillary density—The number ofcollateral capillaries determined in light microscopic sections takenfrom hindlimbs.

[1087] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 48 Effect of Polypeptides of the Invention on Vasodilation

[1088] Since dilation of vascular endothelium is important in reducingblood pressure, the ability of polypeptides of the invention to affectthe blood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as themean+/−SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

[1089] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 49 Rat Ischemic Skin Flap Model

[1090] The evaluation parameters include skin blood flow, skintemperature, and factor VIII immunohistochemistry or endothelialalkaline phosphatase reaction. Expression of polypeptides of theinvention, during the skin ischemia, is studied using in situhybridization. The study in this model is divided into three parts asfollows:

[1091] a) Ischemic skin

[1092] b) Ischemic skin wounds

[1093] c) Normal wounds

[1094] The experimental protocol includes:

[1095] a) Raising a 3×4 cm, single pedicle full-thickness random skinflap (myocutaneous flap over the lower back of the animal).

[1096] b) An excisional wounding (4-6 mm in diameter) in the ischemicskin (skin-flap).

[1097] c) Topical treatment with a polypeptide of the invention of theexcisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the followingvarious dosage ranges: 1 mg to 100 mg.

[1098] d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

[1099] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 50 Peripheral Arterial Disease Model

[1100] Angiogenic therapy using a polypeptide of the invention is anovel therapeutic strategy to obtain restoration of blood flow aroundthe ischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

[1101] a) One side of the femoral artery is ligated to create ischemicmuscle of the hindlimb, the other side of hindlimb serves as a control.

[1102] b) a polypeptide of the invention, in a dosage range of 20 mg-500mg, is delivered intravenously and/or intramuscularly 3 times (perhapsmore) per week for 2-3 weeks.

[1103] c) The ischemic muscle tissue is collected after ligation of thefemoral

[1104] artery at 1, 2, and 3 weeks for the analysis of expression of apolypeptide of the invention and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

[1105] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 51 Ischemic Myocardial Disease Model

[1106] A polypeptide of the invention is evaluated as a potent mitogencapable of stimulating the development of collateral vessels, andrestructuring new vessels after coronary artery occlusion. Alteration ofexpression of the polypeptide is investigated in situ. The experimentalprotocol includes:

[1107] a) The heart is exposed through a left-side thoracotomy in therat. Immediately, the left coronary artery is occluded with a thinsuture (6-0) and the thorax is closed.

[1108] b) a polypeptide of the invention, in a dosage range of 20 mg-500mg, is delivered intravenously and/or intramuscularly 3 times (perhapsmore) per week for 2-4 weeks.

[1109] c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

[1110] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 52 Rat Corneal Wound Healing Model

[1111] This animal model shows the effect of a polypeptide of theinvention on neovascularization. The experimental protocol includes:

[1112] a) Making a 1-1.5 mm long incision from the center of cornea intothe stromal layer.

[1113] b) Inserting a spatula below the lip of the incision facing theouter corner of the eye.

[1114] c) Making a pocket (its base is 1-1.5 mm form the edge of theeye).

[1115] d) Positioning a pellet, containing 50 ng-5 ug of a polypeptideof the invention, within the pocket.

[1116] e) Treatment with a polypeptide of the invention can also beapplied topically to the corneal wounds in a dosage range of 20 mg-500mg (daily treatment for five days).

[1117] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 53 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

[1118] A. Diabetic db+/db+ Mouse Model.

[1119] To demonstrate that a polypeptide of the invention acceleratesthe healing process, the genetically diabetic mouse model of woundhealing is used. The full thickness wound healing model in the db+/db+mouse is a well characterized, clinically relevant and reproduciblemodel of impaired wound healing. Healing of the diabetic wound isdependent on formation of granulation tissue and re-epithelializationrather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389(1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

[1120] The diabetic animals have many of the characteristic featuresobserved in Type II diabetes mellitus. Homozygous (db+/db+) mice areobese in comparison to their normal heterozygous (db+/+m) littermates.Mutant diabetic (db+/db+) mice have a single autosomal recessivemutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci.USA 77:283-293 (1982)). Animals show polyphagia, polydipsia andpolyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose,increased or normal insulin levels, and suppressed cell-mediatedimmunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M.et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. ofPathol. 114:46-55 (1985)). Peripheral neuropathy, myocardialcomplications, and microvascular lesions, basement membrane thickeningand glomerular filtration abnormalities have been described in theseanimals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertsonet al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)).These homozygous diabetic mice develop hyperglycemia that is resistantto insulin analogous to human type II diabetes (Mandel et al., J.Immunol. 120:1375-1377 (1978)).

[1121] The characteristics observed in these animals suggests thathealing in this model may be similar to the healing observed in humandiabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

[1122] Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Bristol-Myers Squibb Company'sInstitutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

[1123] Wounding protocol is performed according to previously reportedmethods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

[1124] Wounds are visually examined and photographed at a fixed distanceat the day of surgery and at two day intervals thereafter. Wound closureis determined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[1125] A polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

[1126] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

[1127] Three groups of 10 animals each (5 diabetic and 5 non-diabeticcontrols) are evaluated: 1) Vehicle placebo control, 2) untreated group,and 3) treated group.

[1128] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm2, the corresponding size of the dermalpunch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[1129] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with a polypeptide of the invention. This assessment includedverification of the presence of cell accumulation, inflammatory cells,capillaries, fibroblasts, re-epithelialization and epidermal maturity(Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibratedlens micrometer is used by a blinded observer.

[1130] Tissue sections are also stained immunohistochemically with apolyclonal rabbit anti-human keratin antibody using ABC Elite detectionsystem. Human skin is used as a positive tissue control while non-immuneIgG is used as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

[1131] Proliferating cell nuclear antigen/cyclin (PCNA) in skinspecimens is demonstrated by using anti-PCNA antibody (1:50) with an ABCElite detection system. Human colon cancer can serve as a positivetissue control and human brain tissue can be used as a negative tissuecontrol. Each specimen includes a section with omission of the primaryantibody and substitution with non-immune mouse IgG. Ranking of thesesections is based on the extent of proliferation on a scale of 0-8, thelower side of the scale reflecting slight proliferation to the higherside reflecting intense proliferation.

[1132] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[1133] B. Steroid Impaired Rat Model

[1134] The inhibition of wound healing by steroids has been welldocumented in various in vitro and in vivo systems (Wahl,Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action:Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115:476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin.Invest. 61: 703-797 (1978)) and producing a transient reduction ofcirculating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797(1978); Wahl, “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

[1135] To demonstrate that a polypeptide of the invention can acceleratethe healing process, the effects of multiple topical applications of thepolypeptide on full thickness excisional skin wounds in rats in whichhealing has been impaired by the systemic administration ofmethylprednisolone is assessed.

[1136] Young adult male Sprague Dawley rats weighing 250-300 g (CharlesRiver Laboratories) are used in this example. The animals are purchasedat 8 weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy would be conducted according to the rules and guidelines ofBristol-Myers Squibb Corporations Guidelines for the Care and Use ofLaboratory Animals.

[1137] The wounding protocol is followed according to section A, above.On the day of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

[1138] Wounds are visually examined and photographed at a fixed distanceat the day of wounding and at the end of treatment. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[1139] The polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

[1140] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

[1141] Four groups of 10 animals each (5 with methylprednisolone and 5without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicleplacebo control 3) treated groups.

[1142] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total area of the wound. Closureis then estimated by establishing the differences between the initialwound area (day 0) and that of post treatment (day 8). The wound area onday 1 is 64 mm2, the corresponding size of the dermal punch.Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[1143] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds allows assessment of whether the healingprocess and the morphologic appearance of the repaired skin is improvedby treatment with a polypeptide of the invention. A calibrated lensmicrometer is used by a blinded observer to determine the distance ofthe wound gap.

[1144] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[1145] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 54 Lymphedema Animal Model

[1146] The purpose of this experimental approach is to create anappropriate and consistent lymphedema model for testing the therapeuticeffects of a polypeptide of the invention in lymphangiogenesis andre-establishment of the lymphatic circulatory system in the rat hindlimb. Effectiveness is measured by swelling volume of the affected limb,quantification of the amount of lymphatic vasculature, total bloodplasma protein, and histopathology. Acute lymphedema is observed for7-10 days. Perhaps more importantly, the chronic progress of the edemais followed for up to 3-4 weeks.

[1147] Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

[1148] Using the knee joint as a landmark, a mid-leg inguinal incisionis made circumferentially allowing the femoral vessels to be located.Forceps and hemostats are used to dissect and separate the skin flaps.After locating the femoral vessels, the lymphatic vessel that runs alongside and underneath the vessel(s) is located. The main lymphatic vesselsin this area are then electrically coagulated suture ligated.

[1149] Using a microscope, muscles in back of the leg (near thesemitendinosis and adductors) are bluntly dissected. The popliteal lymphnode is then located. The 2 proximal and 2 distal lymphatic vessels anddistal blood supply of the popliteal node are then and ligated bysuturing. The popliteal lymph node, and any accompanying adipose tissue,is then removed by cutting connective tissues.

[1150] Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

[1151] To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

[1152] Circumference Measurements: Under brief gas anesthetic to preventlimb movement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

[1153] Volumetric Measurements: On the day of surgery, animals areanesthetized with Pentobarbital and are tested prior to surgery. Fordaily volumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

[1154] Blood-plasma protein measurements: Blood is drawn, spun, andserum separated prior to surgery and then at conclusion for totalprotein and Ca2+ comparison.

[1155] Limb Weight Comparison: After drawing blood, the animal isprepared for tissue collection. The limbs are amputated using aquillitine, then both experimental and control legs are cut at theligature and weighed. A second weighing is done as the tibio-cacanealjoint is disarticulated and the foot is weighed.

[1156] Histological Preparations: The transverse muscle located behindthe knee (popliteal) area is dissected and arranged in a metal mold,filled with freezeGel, dipped into cold methylbutane, placed intolabeled sample bags at −80EC until sectioning. Upon sectioning, themuscle is observed under fluorescent microscopy for lymphatics.

[1157] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 55 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby a Polypeptide of the Invention

[1158] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[1159] Tumor necrosis factor alpha (TNF-a), a potent proinflammatorycytokine, is a stimulator of all three CAMs on endothelial cells and maybe involved in a wide variety of inflammatory responses, often resultingin a pathological outcome.

[1160] The potential of a polypeptide of the invention to mediate asuppression of TNF-a induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-a treated ECs whenco-stimulated with a member of the FGF family of proteins.

[1161] To perform the experiment, human umbilical vein endothelial cell(HUVEC) cultures are obtained from pooled cord harvests and maintainedin growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidifiedincubator containing 5% CO₂. HUVECs are seeded in 96-well plates atconcentrations of 1×104 cells/well in EGM medium at 37 degree C. for18-24 hrs or until confluent. The monolayers are subsequently washed 3times with a serum-free solution of RPMI-1640 supplemented with 100 U/mlpenicillin and 100 mg/ml streptomycin, and treated with a given cytokineand/or growth factor(s) for 24 h at 37 degree C. Following incubation,the cells are then evaluated for CAM expression.

[1162] Human Umbilical Vein Endothelial cells (HUVECs) are grown in astandard 96 well plate to confluence. Growth medium is removed from thecells and replaced with 90 ul of 199 Medium (10% FBS). Samples fortesting and positive or negative controls are added to the plate intriplicate (in 10 ul volumes). Plates are incubated at 37 degree C. foreither 5 h (selectin and integrin expression) or 24 h (integrinexpression only). Plates are aspirated to remove medium and 100 μl of0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well.Plates are held at 4° C. for 30 min.

[1163] Fixative is then removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry.Add 10 μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

[1164] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase(1:5,000 dilution) to each well and incubated at 37° C. for 30 min.Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000(100)>10-0.5>10-1>10-1.5. 5 μl of each dilution is added to triplicatewells and the resulting AP content in each well is 5.50 ng, 1.74 ng,0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each ofthe standard wells. The plate must be incubated at 37° C. for 4 h. Avolume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

[1165] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 56 Method of Creating N- and C-Terminal Deletion Mutants.Corresponding to the HLRRSI1 Polypeptide of the Present Invention

[1166] As described elsewhere herein, the present invention encompassesthe creation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to theHLRRSI1 polypeptide of the present invention. A number of methods areavailable to one skilled in the art for creating such mutants. Suchmethods may include a combination of PCR amplification and gene cloningmethodology. Although one of skill in the art of molecular biology,through the use of the teachings provided or referenced herein, and/orotherwise known in the art as standard methods, could readily createeach deletion mutant of the present invention, exemplary methods aredescribed below.

[1167] Briefly, using the isolated cDNA clone encoding the full-lengthHLRRSI1 polypeptide sequence (as described in Example 12, for example),appropriate primers of about 15-25 nucleotides derived from the desired5′ and 3′ positions of SEQ ID NO:1 may be designed to PCR amplify, andsubsequently clone, the intended N- and/or C-terminal deletion mutant.Such primers could comprise, for example, an inititation and stop codonfor the 5′ and 3′ primer, respectively. Such primers may also compriserestriction sites to facilitate cloning of the deletion mutant postamplification. Moreover, the primers may comprise additional sequences,such as, for example, flag-tag sequences, kozac sequences, or othersequences discussed and/or referenced herein.

[1168] For example, in the case of the D168 to F625 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′-GCAGCA GCGGCCGCGACGGGCCCCGGTTGCAGGGCGACC-3′ (SEQ ID NO:29)              NotI 3′ Primer5′-GCAGCA GTCGAC GAAGGTCGAGATGAGTTCCTTGGG -3′(SEQ ID NO:30)            SalI

[1169] For example, in the case of the M1 to D551 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′-GCAGCA GCGGCCGCATGCTGGCCCAGCCGCAGCGGCTGC-3′ (SEQ ID NO:31)             NotI 3′ Primer5′-GCAGCA GTCGAC ATCCAGGGTGGTCAGGGCGGGGCTC-3′ (SEQ ID NO:32)            SalI

[1170] Representative PCR amplification conditions are provided below,although the skilled artisan would appreciate that other conditions maybe required for efficient amplification. A 100 ul PCR reaction mixturemay be prepared using 10 ng of the template DNA (cDNA clone of HLRRSI1),200 uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standardTaq DNA polymerase buffer. Typical PCR cycling condition are as follows:

[1171] 20-25 cycles: 45 sec, 93 degrees

[1172] 2 min, 50 degrees

[1173] 2 min, 72 degrees

[1174] 1 cycle: 10 min, 72 degrees

[1175] After the final extension step of PCR, 5U Klenow Fragment may beadded and incubated for 15 min at 30 degrees.

[1176] Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

[1177] The 5′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula:

[1178] (S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the HLRRSI1 gene(SEQ ID NO:1), and ‘X’ is equal to the most N-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 5′ primer, while the second termwill provide the end 3′ nucleotide position of the 5′ primercorresponding to sense strand of SEQ ID NO:1. Once the correspondingnucleotide positions of the primer are determined, the final nucleotidesequence may be created by the addition of applicable restriction sitesequences to the 5′ end of the sequence, for example. As referencedherein, the addition of other sequences to the 5′ primer may be desiredin certain circumstances (e.g., kozac sequences, etc.).

[1179] The 3′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula:

[1180] (S+(X*3)) to ((S+(X*3))-25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the HLRRSI1 gene(SEQ ID NO:1), and ‘X’ is equal to the most C-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 3′ primer, while the second termwill provide the end 3′ nucleotide position of the 3′ primercorresponding to the anti-sense strand of SEQ ID NO:1. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 3′ primermay be desired in certain circumstances (e.g., stop codon sequences,etc.). The skilled artisan would appreciate that modifications of theabove nucleotide positions may be necessary for optimizing PCRamplification.

[1181] The same general formulas provided above may be used inidentifying the 5′ and 3′ primer sequences for amplifying any C-terminaldeletion mutant of the present invention. Moreover, the same generalformulas provided above may be used in identifying the 5′ and 3′ primersequences for amplifying any combination of N-terminal and C-terminaldeletion mutant of the present invention. The skilled artisan wouldappreciate that modifications of the above nucleotide positions may benecessary for optimizing PCR amplification.

Example 57 Complementary Polynucleotides

[1182] Antisense molecules or nucleic acid sequences complementary tothe HLRRSI1 protein-encoding sequence, or any part thereof, is used todecrease or to inhibit the expression of naturally occurring HLRRSI1.Although the use of antisense or complementary oligonucleotidescomprising about 15 to 35 base-pairs is described, essentially the sameprocedure is used with smaller or larger nucleic acid sequencefragments. An oligonucleotide based on the coding sequence of HLRRSI1protein, as shown in FIGS. 1A-C, or as depicted in SEQ ID NO:1, forexample, is used to inhibit expression of naturally occurring HLRRSI1.The complementary oligonucleotide is typically designed from the mostunique 5′ sequence and is used either to inhibit transcription bypreventing promoter binding to the coding sequence, or to inhibittranslation by preventing the ribosome from binding to the HLRRSI1protein-encoding transcript, among others. However, other regions mayalso be targeted.

[1183] Using an appropriate portion of the signal and 5′ sequence of SEQID NO:1, an effective antisense oligonucleotide includes any of about15-35 nucleotides spanning the region which translates into the signalor 5′ coding sequence, among other regions, of the polypeptide as shownin FIGS. 1A-C (SEQ ID NO:2). Appropriate oligonucleotides are designedusing OLIGO 4.06 software and the HLRRSI1 protein coding sequence (SEQID NO:1). Preferred oligonucleotides are dideoxy based and are providedbelow. The oligonucleotides were synthesized using chemistry essentiallyas described in U.S. Pat. No. 5,849,902; which is hereby incorporatedherein by reference in its entirety. ID # Sequence 13713CCTCTCATCCCGGAAGAACUUGUAG (SEQ ID NO:34) 13714 GGCCTCCTGCUUCACACGCUCUGAA(SEQ ID NO:35) 13715 AACTCCTGGAAGCUCUGGUCGAUGA (SEQ ID NO:36) 13716GTCTGCACTUUGGAGCCACGAAGCT (SEQ ID NO:37) 13717 TTCTCCTTCACGAAGCGGUAGGCGC(SEQ ID NO:38)

[1184] The HLRRSI1 polypeptide has been shown to be involved in theregulation of mammalian NF-κB and apoptosis pathways. Subjecting cellswith an effective amount of a pool of all five of the above antisenseoligoncleotides resulted in a significant increase in IκBαexpression/activity providing convincing evidence that HLRRSI1 at leastregulates the activity and/or expression of IκBα either directly, orindirectly. Moreover, the results suggest that HLRRSI1 is involved inthe negative regulation of NF-κB/IκBα activity and/or expression, eitherdirectly or indirectly. The IκBα assay used is described below and wasbased upon the analysis of IκBα activity as a downstream marker forproliferative signal transduction events.

[1185] Transfection of Post-Quiescent A549 cells with AntiSenseOligonucleotides.

[1186] Materials needed:

[1187] A549 cells maintained in DMEM with high glucose (Gibco-BRL)supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and 1×penicillin/streptomycin.

[1188] Opti-MEM (Gibco-BRL)

[1189] Lipofectamine 2000 (Invitrogen)

[1190] Antisense oligomers (Sequitur)

[1191] Polystyrene tubes.

[1192] Tissue culture treated plates.

[1193] Quiescent cells were prepared as follows:

[1194] Day 0: 300,000 A549 cells were seeded in a T75 tissue cultureflask in 10 ml of A549 media, and incubated in at 37° C., 5% CO₂ in ahumidified incubator for 48 hours.

[1195] Day 2: The T75 flasks were rocked to remove any loosely adherentcells, and the A549 growth media removed and replenished with 10 ml offresh A549 media. The cells were cultured for six days without changingthe media to create a quiescent cell population.

[1196] Day 8: Quiescent cells were plated in multi-well format andtransfected with antisense oligonucleotides.

[1197] A549 cells were transfected according to the following:

[1198] 1. Trypsinize T75 flask containing quiescent population of A549cells.

[1199] 2. Count the cells and seed 24-well plates with 60K quiescentA549 cells per well.

[1200] 3. Allow the cells to adhere to the tissue culture plate(approximately 4 hours).

[1201] 4. Transfect the cells with antisense and controloligonucleotides according to the following:

[1202] a. A 10× stock of lipofectamine 2000 (10 ug/ml is 10×) wasprepared, and diluted lipid was allowed to stand at RT for 15 minutes.

[1203] Stock solution of lipofectamine 2000 was 1 mg/ml.

[1204] 10× solution for transfection was 10 ug/ml.

[1205] To prepare 10× solution, dilute 10 ul of lipofectamine 2000 stockper 1 ml of Opti-MEM (serum free media).

[1206] b. A 10× stock of each oligomer was prepared to be used in thetransfection.

[1207] Stock solutions of oligomers were at 100 uM in 20 mM HEPES, pH7.5.

[1208] 10× concentration of oligomer was 0.25 uM.

[1209] To prepare the 10× solutions, dilute 2.5 ul of oligomer per 1 mlof Opti-MEM.

[1210] c. Equal volumes of the 10× lipofectamine 2000 stock and the 10×oligomer solutions were mixed well, and incubated for 15 minutes at RTto allow complexation of the oligomer and lipid. The resulting mixturewas 5×.

[1211] d. After the 15 minute complexation, 4 volumes of full growthmedia was added to the oligomer/lipid complexes (solution was 1×).

[1212] e. The media was aspirated from the cells, and 0.5 ml of the 1×oligomer/lipid complexes added to each well.

[1213] f. The cells were incubated for 16-24 hours at 37° C. in ahumidified CO₂ incubator.

[1214] g. Cell pellets were harvested for RNA isolation and TaqMananalysis of downstream marker genes.

[1215] TaqMan Reactions

[1216] Quantitative RT-PCR analysis was performed on total RNA prepsthat had been treated with DNaseI or poly A selected RNA. The Dnasetreatment may be performed using methods known in the art, thoughpreferably using a Qiagen RNeasy kit to purify the RNA samples, whereinDNAse I treatment is performed on the column.

[1217] Briefly, a master mix of reagents was prepared according to thefollowing table: Dnase I Treatment Reagent Per r'xn (in uL) 10x Buffer2.5 Dnase I (1 unit/ul @ 1 unit per ug 2 sample) DEPC H₂O 0.5 RNA sample@ 0.1 ug/ul 20 (2-3 ug total) Total 25

[1218] Next, 5 ul of master mix was aliquoted per well of a 96-well PCRreaction plate (PE part # N801-0560). RNA samples were adjusted to 0.1ug/ul with DEPC treated H₂O (if necessary), and 20 ul was added to thealiquoted master mix for a final reaction volume of 25 ul.

[1219] The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and briefly spun in a centrifuge tocollect all volume in the bottom of the tubes. Generally, a short spinup to 500 rpm in a Sorvall RT is sufficient

[1220] The plates were incubated at 37° C. for 30 mins. Then, an equalvolume of 0.1 mM EDTA in 10 mM Tris was added to each well, and heatinactivated at 70° C. for 5 min. The plates were stored at −80° C. uponcompletion.

[1221] RT Reaction

[1222] A master mix of reagents was prepared according to the followingtable: RT reaction RT No RT Reagent Per Rx'n (in ul) Per Rx'n (in ul)10x RT buffer 5 2.5 MgCl₂ 11 5.5 DNTP mixture 10 5 Random Hexamers 2.51.25 Rnase inhibitors 1.25 0.625 RT enzyme 1.25 — Total RNA 500 ng (100ng no 19.0 max 10.125 max RT) DEPC H₂O — — Total 50 uL 25 uL

[1223] Samples were adjusted to a concentration so that 500 ng of RNAwas added to each RT rx′n (10 ng for the no RT). A maximum of 19 ul canbe added to the RT rx′n mixture (10.125 ul for the no RT.) Any remainingvolume up to the maximum values was filled with DEPC treated H₂O, sothat the total reaction volume was 50 ul (RT) or 25 ul (no RT).

[1224] On a 96-well PCR reaction plate (PE part # N801-0560), 37.5 ul ofmaster mix was aliquoted (22.5 ul of no RT master mix), and the RNAsample added for a total reaction volume of 50 ul (25 ul, no RT).Control samples were loaded into two or even three different wells inorder to have enough template for generation of a standard curve.

[1225] The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and spin briefly in a centrifuge tocollect all volume in the bottom of the tubes. Generally, a short spinup to 500 rpm in a Sorvall RT is sufficient.

[1226] For the RT-PCR reaction, the following thermal profile was used:

[1227] 25° C. for 10 min

[1228] 48° C. for 30 min

[1229] 95° C. for 5 min

[1230] 4° C. hold (for 1 hour)

[1231] Store plate @-20° C. or lower upon completion.

[1232] TaqMan Reaction (Template comes from RT Plate.)

[1233] A master mix was prepared according to the following table:TaqMan reaction (per well) Reagent Per Rx'n (in ul) TaqMan Master Mix4.17 100 uM Probe .025 (SEQ ID NO: 41) 100 uM Forward .05 primer (SEQ IDNO: 39) 100 uM Reverse .05 primer (SEQ ID NO: 40) Template — DEPC H₂O18.21 Total 22.5

[1234] The primers used for the RT-PCR reaction is as follows:

[1235] IκBα primer and probes: Forward Primer: GAGGATGAGGAGAGCTATGACACA(SEQ ID NO:39)

[1236] Anneals between residues 558 and 577 with a Tm of 59°. ReversePrimer: CCCTTTGCACTCATAACGTCAG (SEQ ID NO:40)

[1237] Anneals between residues 639 and 619 with a Tm of 60°. TaqManProbe: AAACACACAGTCATCATAGGGCAGCTCGT (SEQ ID NO:41)

[1238] Anneals between residues 579 and 600 with a Tm of 68°.

[1239] Using a Gilson P-10 repeat pipetter, 22.5 ul of master mix wasaliquouted per well of a 96-well optical plate. Then, using P-10pipetter, 2.5 ul of sample was added to individual wells. Generally, RTsamples are run in triplicate with each primer/probe set used, and no RTsamples are run once and only with one primer/probe set, often gapdh (orother internal control).

[1240] A standard curve is then constructed and loaded onto the plate.The curve has five points plus one no template control (NTC, =DEPCtreated H₂O). The curve was made with a high point of 50 ng of sample(twice the amount of RNA in unknowns), and successive samples of 25, 10,5, and 1 ng. The curve was made from a control sample(s) (see above).

[1241] The wells were capped using optical strip well caps (PE part #N801-0935), placed in a plate, and spun in a centrifuge to collect allvolume in the bottom of the tubes. Generally, a short spin up to 500 rpmin a Sorvall RT is sufficient.

[1242] Plates were loaded onto a PE 5700 sequence detector making surethe plate is aligned properly with the notch in the upper right handcorner. The lid was tightened down and run using the 5700 and 5700quantitation program and the SYBR probe using the following thermalprofile:

[1243] 50° C. for 2 min

[1244] 95° C. for 10 min

[1245] and the following for 40 cycles:

[1246] 95° C. for 15 sec

[1247] 60° C. for 1 min

[1248] Change the reaction volume to 25 ul.

[1249] Once the reaction was complete, a manual threshold of around 0.1was set to minimuze the background signal. Additional informationrelative to operation of the GeneAmp 5700 machine may be found inreference to the following manuals: “GeneAmp 5700 Sequence DetectionSystem Operator Training CD”; and the “User's Manual for 5700 SequenceDetection System”; available from Perkin-Elmer and hereby incorporatedby reference herein in their entirety.

[1250] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[1251] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties.

1 45 1 2689 DNA homo sapiens CDS (75)..(1949) 1 cggacgcgtg ggcgcgcagcctggctgacc tgatcctgga ccagtgcccc gaccgcggcg 60 cgccggtgcc gcag atg ctggcc cag ccg cag cgg ctg ctc ttc atc ctg 110 Met Leu Ala Gln Pro Gln ArgLeu Leu Phe Ile Leu 1 5 10 gac ggc gcg gac gag ctg ccg gcg ctg ggg ggcccc gag gcc gcg ccc 158 Asp Gly Ala Asp Glu Leu Pro Ala Leu Gly Gly ProGlu Ala Ala Pro 15 20 25 tgc aca gac ccc ttc gag gcg gcg agc ggc gcg cgggtg cta ggc ggg 206 Cys Thr Asp Pro Phe Glu Ala Ala Ser Gly Ala Arg ValLeu Gly Gly 30 35 40 ctg ctg agt aag gcg ctg ctg ccc acg gcc ctc ctg ctggtg acc acg 254 Leu Leu Ser Lys Ala Leu Leu Pro Thr Ala Leu Leu Leu ValThr Thr 45 50 55 60 cgc gcc gcc gcc ccc ggg agg ctg cag ggc cgc ctg tgttcc ccg cag 302 Arg Ala Ala Ala Pro Gly Arg Leu Gln Gly Arg Leu Cys SerPro Gln 65 70 75 tgc gcc gag gtg cgc ggc ttc tcc gac aag gac aag aag aagtat ttc 350 Cys Ala Glu Val Arg Gly Phe Ser Asp Lys Asp Lys Lys Lys TyrPhe 80 85 90 tac aag ttc ttc cgg gat gag agg agg gcc gag cgc gcc tac cgcttc 398 Tyr Lys Phe Phe Arg Asp Glu Arg Arg Ala Glu Arg Ala Tyr Arg Phe95 100 105 gtg aag gag aac gag acg ctg ttc gcg ctg tgc ttc gtg ccc ttcgtg 446 Val Lys Glu Asn Glu Thr Leu Phe Ala Leu Cys Phe Val Pro Phe Val110 115 120 tgc tgg atc gtg tgc acc gtg ctg cgc cag cag ctg gag ctc ggtcgg 494 Cys Trp Ile Val Cys Thr Val Leu Arg Gln Gln Leu Glu Leu Gly Arg125 130 135 140 gac ctg tcg cgc acg tcc aag acc acc acg tca gtg tac ctgctt ttc 542 Asp Leu Ser Arg Thr Ser Lys Thr Thr Thr Ser Val Tyr Leu LeuPhe 145 150 155 atc acc agc gtt ctg agc tcg gct ccg gta gcc gac ggg ccccgg ttg 590 Ile Thr Ser Val Leu Ser Ser Ala Pro Val Ala Asp Gly Pro ArgLeu 160 165 170 cag ggc gac ctg cgc aat ctg tgc cgc ctg gcc cgc gag ggcgtc ctc 638 Gln Gly Asp Leu Arg Asn Leu Cys Arg Leu Ala Arg Glu Gly ValLeu 175 180 185 gga cgc agg gcg cag ttt gcc gag aag gaa ctg gag caa ctggag ctt 686 Gly Arg Arg Ala Gln Phe Ala Glu Lys Glu Leu Glu Gln Leu GluLeu 190 195 200 cgt ggc tcc aaa gtg cag acg ctg ttt ctc agc aaa aag gagctg ccg 734 Arg Gly Ser Lys Val Gln Thr Leu Phe Leu Ser Lys Lys Glu LeuPro 205 210 215 220 ggc gtg ctg gag aca gag gtc acc tac cag ttc atc gaccag agc ttc 782 Gly Val Leu Glu Thr Glu Val Thr Tyr Gln Phe Ile Asp GlnSer Phe 225 230 235 cag gag ttc ctc gcg gca ctg tcc tac ctg ctg gag gacggc ggg gtg 830 Gln Glu Phe Leu Ala Ala Leu Ser Tyr Leu Leu Glu Asp GlyGly Val 240 245 250 ccc agg acc gcg gct ggc ggc gtt ggg aca ctc ctg cgtggg gac gcc 878 Pro Arg Thr Ala Ala Gly Gly Val Gly Thr Leu Leu Arg GlyAsp Ala 255 260 265 cag ccg cac agc cac ttg gtg ctc acc acg cgc ttc ctcttc gga ctg 926 Gln Pro His Ser His Leu Val Leu Thr Thr Arg Phe Leu PheGly Leu 270 275 280 ctg agc gcg gag cgg atg cgc gac atc gag cgc cac ttcggc tgc atg 974 Leu Ser Ala Glu Arg Met Arg Asp Ile Glu Arg His Phe GlyCys Met 285 290 295 300 gtt tca gag cgt gtg aag cag gag gcc ctg cgg tgggtg cag gga cag 1022 Val Ser Glu Arg Val Lys Gln Glu Ala Leu Arg Trp ValGln Gly Gln 305 310 315 gga cag ggc tgc ccc gga gtg gca cca gag gtg accgag ggg gcc aaa 1070 Gly Gln Gly Cys Pro Gly Val Ala Pro Glu Val Thr GluGly Ala Lys 320 325 330 ggg ctc gag gac acc gaa gag cca gag gag gag gaggag gga gag gag 1118 Gly Leu Glu Asp Thr Glu Glu Pro Glu Glu Glu Glu GluGly Glu Glu 335 340 345 ccc aac tac cca ctg gag ttg ctg tac tgc ctg tacgag acg cag gag 1166 Pro Asn Tyr Pro Leu Glu Leu Leu Tyr Cys Leu Tyr GluThr Gln Glu 350 355 360 gac gcg ttt gtg cgc caa gcc ctg tgc cgg ttc ccggag ctg gcg ctg 1214 Asp Ala Phe Val Arg Gln Ala Leu Cys Arg Phe Pro GluLeu Ala Leu 365 370 375 380 cag cga gtg cgc ttc tgc cgc atg gac gtg gctgtt ctg agc tac tgc 1262 Gln Arg Val Arg Phe Cys Arg Met Asp Val Ala ValLeu Ser Tyr Cys 385 390 395 gtg agg tgc tgc cct gct gga cag gca ctg cggctg atc agc tgc aga 1310 Val Arg Cys Cys Pro Ala Gly Gln Ala Leu Arg LeuIle Ser Cys Arg 400 405 410 ttg gtt gct gcg cag gag aag aag aag aag agcctg ggg aag cgg ctc 1358 Leu Val Ala Ala Gln Glu Lys Lys Lys Lys Ser LeuGly Lys Arg Leu 415 420 425 cag gcc agc ctg ggt ggc ggc agt tct caa ggcacc aca aaa caa ctg 1406 Gln Ala Ser Leu Gly Gly Gly Ser Ser Gln Gly ThrThr Lys Gln Leu 430 435 440 cca gcc tcc ctt ctt cat cca ctc ttt cag gcaatg act gac cca ctg 1454 Pro Ala Ser Leu Leu His Pro Leu Phe Gln Ala MetThr Asp Pro Leu 445 450 455 460 tgc cat ctg agc agc ctc acg ctg tcc cactgc aaa ctc cct gac gcg 1502 Cys His Leu Ser Ser Leu Thr Leu Ser His CysLys Leu Pro Asp Ala 465 470 475 gtc tgc cga gac ctt tct gag gcc ctg agggca gcc ccc gca ctg acg 1550 Val Cys Arg Asp Leu Ser Glu Ala Leu Arg AlaAla Pro Ala Leu Thr 480 485 490 gag ctg ggc ctc ctc cac aac agg ctc agtgag gcg gga ctg cgt atg 1598 Glu Leu Gly Leu Leu His Asn Arg Leu Ser GluAla Gly Leu Arg Met 495 500 505 ctg agt gag ggc cta gcc tgg ccg cag tgcagg gtg cag acg gtc agg 1646 Leu Ser Glu Gly Leu Ala Trp Pro Gln Cys ArgVal Gln Thr Val Arg 510 515 520 gta cag ctg cct gac ccc cag cga ggg ctccag tac ctg gtg ggt atg 1694 Val Gln Leu Pro Asp Pro Gln Arg Gly Leu GlnTyr Leu Val Gly Met 525 530 535 540 ctt cgg cag agc ccc gcc ctg acc accctg gat ctc agc ggc tgc caa 1742 Leu Arg Gln Ser Pro Ala Leu Thr Thr LeuAsp Leu Ser Gly Cys Gln 545 550 555 ctg ccc gcc ccc atg gtg acc tac ctgtgt gca gtc ctg cag cac cag 1790 Leu Pro Ala Pro Met Val Thr Tyr Leu CysAla Val Leu Gln His Gln 560 565 570 gga tgc ggc ctg cag acc ctc agt ctggcc tct gtg gag ctg agc gag 1838 Gly Cys Gly Leu Gln Thr Leu Ser Leu AlaSer Val Glu Leu Ser Glu 575 580 585 cag tca cta cag gag ctt cag gct gtgaag aga gca aag ccg gat ctg 1886 Gln Ser Leu Gln Glu Leu Gln Ala Val LysArg Ala Lys Pro Asp Leu 590 595 600 gtc atc aca cac cca gcg ctg gac ggccac cca caa cct ccc aag gaa 1934 Val Ile Thr His Pro Ala Leu Asp Gly HisPro Gln Pro Pro Lys Glu 605 610 615 620 ctc atc tcg acc ttc tgaggctctggtggccagag cagggtggaa gaccctagtc 1989 Leu Ile Ser Thr Phe 625 aaagtccctgtggagagaac ggcccattcc aagggcagga ggatattgct ctcggccttt 2049 gggaaacttttgagccgaga ggccgcagac aggcatgtgg gaggcccaga cacggcaccc 2109 tgccccgtccaggacaggcc caggacctgc ccctctctcc acacctgggg taccccttct 2169 cccccagccccaccactact ccacccacct tcctctcctg agaccctcca gccattcccc 2229 ttgaaaacaccccccgaccc caagccacaa taatgacagc gagagctcca attaactaag 2289 cacctacctggcggcagaat aacccttcac tgcctgatcc ccatctgcag tgtggcccaa 2349 cagcccccagaactatgccc acatagactg gaggtaggca gttcaccgtc cctccctgtt 2409 aggaatgagaccatccctga ggctatggcc caggcccaca ggcgtccagt gtctgagatc 2469 tttgggaagggagactaggg caggtggaga cagcgcagaa cccccgtgct gggtgggaag 2529 catgaccacatggtgggtga gcagccccca tgcactgacg gtaaattccc ctgtggactc 2589 atttctgttggtttctatta cacctggcca ggcgtggtac aatacaggtc ggtgctcaca 2649 aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2689 2 625 PRT homo sapiens 2 Met LeuAla Gln Pro Gln Arg Leu Leu Phe Ile Leu Asp Gly Ala Asp 1 5 10 15 GluLeu Pro Ala Leu Gly Gly Pro Glu Ala Ala Pro Cys Thr Asp Pro 20 25 30 PheGlu Ala Ala Ser Gly Ala Arg Val Leu Gly Gly Leu Leu Ser Lys 35 40 45 AlaLeu Leu Pro Thr Ala Leu Leu Leu Val Thr Thr Arg Ala Ala Ala 50 55 60 ProGly Arg Leu Gln Gly Arg Leu Cys Ser Pro Gln Cys Ala Glu Val 65 70 75 80Arg Gly Phe Ser Asp Lys Asp Lys Lys Lys Tyr Phe Tyr Lys Phe Phe 85 90 95Arg Asp Glu Arg Arg Ala Glu Arg Ala Tyr Arg Phe Val Lys Glu Asn 100 105110 Glu Thr Leu Phe Ala Leu Cys Phe Val Pro Phe Val Cys Trp Ile Val 115120 125 Cys Thr Val Leu Arg Gln Gln Leu Glu Leu Gly Arg Asp Leu Ser Arg130 135 140 Thr Ser Lys Thr Thr Thr Ser Val Tyr Leu Leu Phe Ile Thr SerVal 145 150 155 160 Leu Ser Ser Ala Pro Val Ala Asp Gly Pro Arg Leu GlnGly Asp Leu 165 170 175 Arg Asn Leu Cys Arg Leu Ala Arg Glu Gly Val LeuGly Arg Arg Ala 180 185 190 Gln Phe Ala Glu Lys Glu Leu Glu Gln Leu GluLeu Arg Gly Ser Lys 195 200 205 Val Gln Thr Leu Phe Leu Ser Lys Lys GluLeu Pro Gly Val Leu Glu 210 215 220 Thr Glu Val Thr Tyr Gln Phe Ile AspGln Ser Phe Gln Glu Phe Leu 225 230 235 240 Ala Ala Leu Ser Tyr Leu LeuGlu Asp Gly Gly Val Pro Arg Thr Ala 245 250 255 Ala Gly Gly Val Gly ThrLeu Leu Arg Gly Asp Ala Gln Pro His Ser 260 265 270 His Leu Val Leu ThrThr Arg Phe Leu Phe Gly Leu Leu Ser Ala Glu 275 280 285 Arg Met Arg AspIle Glu Arg His Phe Gly Cys Met Val Ser Glu Arg 290 295 300 Val Lys GlnGlu Ala Leu Arg Trp Val Gln Gly Gln Gly Gln Gly Cys 305 310 315 320 ProGly Val Ala Pro Glu Val Thr Glu Gly Ala Lys Gly Leu Glu Asp 325 330 335Thr Glu Glu Pro Glu Glu Glu Glu Glu Gly Glu Glu Pro Asn Tyr Pro 340 345350 Leu Glu Leu Leu Tyr Cys Leu Tyr Glu Thr Gln Glu Asp Ala Phe Val 355360 365 Arg Gln Ala Leu Cys Arg Phe Pro Glu Leu Ala Leu Gln Arg Val Arg370 375 380 Phe Cys Arg Met Asp Val Ala Val Leu Ser Tyr Cys Val Arg CysCys 385 390 395 400 Pro Ala Gly Gln Ala Leu Arg Leu Ile Ser Cys Arg LeuVal Ala Ala 405 410 415 Gln Glu Lys Lys Lys Lys Ser Leu Gly Lys Arg LeuGln Ala Ser Leu 420 425 430 Gly Gly Gly Ser Ser Gln Gly Thr Thr Lys GlnLeu Pro Ala Ser Leu 435 440 445 Leu His Pro Leu Phe Gln Ala Met Thr AspPro Leu Cys His Leu Ser 450 455 460 Ser Leu Thr Leu Ser His Cys Lys LeuPro Asp Ala Val Cys Arg Asp 465 470 475 480 Leu Ser Glu Ala Leu Arg AlaAla Pro Ala Leu Thr Glu Leu Gly Leu 485 490 495 Leu His Asn Arg Leu SerGlu Ala Gly Leu Arg Met Leu Ser Glu Gly 500 505 510 Leu Ala Trp Pro GlnCys Arg Val Gln Thr Val Arg Val Gln Leu Pro 515 520 525 Asp Pro Gln ArgGly Leu Gln Tyr Leu Val Gly Met Leu Arg Gln Ser 530 535 540 Pro Ala LeuThr Thr Leu Asp Leu Ser Gly Cys Gln Leu Pro Ala Pro 545 550 555 560 MetVal Thr Tyr Leu Cys Ala Val Leu Gln His Gln Gly Cys Gly Leu 565 570 575Gln Thr Leu Ser Leu Ala Ser Val Glu Leu Ser Glu Gln Ser Leu Gln 580 585590 Glu Leu Gln Ala Val Lys Arg Ala Lys Pro Asp Leu Val Ile Thr His 595600 605 Pro Ala Leu Asp Gly His Pro Gln Pro Pro Lys Glu Leu Ile Ser Thr610 615 620 Phe 625 3 1429 PRT homo sapiens 3 Met Ala Gly Gly Ala TrpGly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15 Lys Lys Glu Glu LeuLys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30 His Ser Arg Ser SerSer Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45 Ser Gly Met Glu ValAla Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60 Arg Ala Trp Asp LeuAla Leu His Thr Trp Glu Gln Met Gly Leu Arg 65 70 75 80 Ser Leu Cys AlaGln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 85 90 95 Pro Tyr Ser ProSer Glu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100 105 110 Ser Thr AlaVal Leu Met Pro Trp Ile His Glu Leu Pro Ala Gly Cys 115 120 125 Thr GlnGly Ser Glu Arg Arg Val Leu Arg Gln Leu Pro Asp Thr Ser 130 135 140 GlyArg Arg Trp Arg Glu Ile Ser Ala Ser His Leu Tyr Gln Ala Leu 145 150 155160 Pro Ser Ser Pro Asp His Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala 165170 175 Pro Thr Ser Thr Ala Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro180 185 190 Ser Leu Ala Pro Arg Glu Gln Glu Ala Pro Gly Thr Gln Trp ProLeu 195 200 205 Asp Glu Thr Ser Gly Ile Tyr Tyr Thr Glu Ile Arg Glu ArgGlu Arg 210 215 220 Glu Lys Ser Glu Lys Gly Arg Pro Pro Trp Ala Ala ValVal Gly Thr 225 230 235 240 Pro Pro Gln Ala His Ser Ser Leu Gln Pro HisHis His Pro Trp Glu 245 250 255 Pro Ser Val Arg Glu Ser Leu Cys Ser ThrTrp Pro Trp Lys Asn Glu 260 265 270 Asp Phe Asn Gln Lys Phe Thr Gln LeuLeu Leu Leu Gln Arg Pro His 275 280 285 Pro Arg Ser Gln Asp Pro Leu ValLys Arg Ser Trp Pro Asp Tyr Val 290 295 300 Glu Glu Asn Arg Gly His LeuIle Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315 320 Gly Leu Asp Thr GlnGlu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330 335 Gly Ile Gly LysSer Thr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340 345 350 Arg Gly GlnLeu Tyr Gly Asp Arg Phe Gln His Val Phe Tyr Phe Ser 355 360 365 Cys ArgGlu Leu Ala Gln Ser Lys Val Val Ser Leu Ala Glu Leu Ile 370 375 380 GlyLys Asp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln Ile Leu Ser 385 390 395400 Arg Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405410 415 Trp Val Leu Gln Glu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln420 425 430 Pro Gln Pro Ala Asp Ala Leu Leu Gly Ser Leu Leu Gly Lys ThrIle 435 440 445 Leu Pro Glu Ala Ser Phe Leu Ile Thr Ala Arg Thr Thr AlaLeu Gln 450 455 460 Asn Leu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val GluVal Leu Gly 465 470 475 480 Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe TyrArg Tyr Phe Thr Asp 485 490 495 Glu Arg Gln Ala Ile Arg Ala Phe Arg LeuVal Lys Ser Asn Lys Glu 500 505 510 Leu Trp Ala Leu Cys Leu Val Pro TrpVal Ser Trp Leu Ala Cys Thr 515 520 525 Cys Leu Met Gln Gln Met Lys ArgLys Glu Lys Leu Thr Leu Thr Ser 530 535 540 Lys Thr Thr Thr Thr Leu CysLeu His Tyr Leu Ala Gln Ala Leu Gln 545 550 555 560 Ala Gln Pro Leu GlyPro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565 570 575 Glu Gly Ile TrpGln Lys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580 585 590 Lys His GlyLeu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly 595 600 605 Ile LeuGln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile His Leu 610 615 620 CysPhe Gln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu Glu Asp Glu 625 630 635640 Lys Gly Arg Gly Lys His Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr 645650 655 Leu Glu Ala Tyr Gly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg660 665 670 Phe Leu Leu Gly Leu Leu Ser Asp Glu Gly Glu Arg Glu Met GluAsn 675 680 685 Ile Phe His Cys Arg Leu Ser Gln Gly Arg Asn Leu Met GlnTrp Val 690 695 700 Pro Ser Leu Gln Leu Leu Leu Gln Pro His Ser Leu GluSer Leu His 705 710 715 720 Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe LeuThr Gln Val Met Ala 725 730 735 His Phe Glu Glu Met Gly Met Cys Val GluThr Asp Met Glu Leu Leu 740 745 750 Val Cys Thr Phe Cys Ile Lys Phe SerArg His Val Lys Lys Leu Gln 755 760 765 Leu Ile Glu Gly Arg Gln His ArgSer Thr Trp Ser Pro Ser Met Val 770 775 780 Val Leu Phe Arg Trp Val ProVal Thr Asp Ala Tyr Trp Gln Ile Leu 785 790 795 800 Phe Ser Val Leu LysVal Thr Arg Asn Leu Lys Glu Leu Asp Leu Ser 805 810 815 Gly Asn Ser LeuSer His Ser Ala Val Lys Ser Leu Cys Lys Thr Leu 820 825 830 Arg Arg ProArg Cys Leu Leu Glu Thr Leu Arg Leu Ala Gly Cys Gly 835 840 845 Leu ThrAla Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu Arg Ala Asn 850 855 860 GlnThr Leu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu Met Asp Ala 865 870 875880 Gly Ala Lys His Leu Cys Gln Arg Leu Arg Gln Pro Ser Cys Lys Leu 885890 895 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu Thr Ser Asp Cys Cys Gln900 905 910 Asp Leu Ala Ser Val Leu Ser Ala Ser Pro Ser Leu Lys Glu LeuAsp 915 920 925 Leu Gln Gln Asn Asn Leu Asp Asp Val Gly Val Arg Leu LeuCys Glu 930 935 940 Gly Leu Arg His Pro Ala Cys Lys Leu Ile Arg Leu GlyLeu Asp Gln 945 950 955 960 Thr Thr Leu Ser Asp Glu Met Arg Gln Glu LeuArg Ala Leu Glu Gln 965 970 975 Glu Lys Pro Gln Leu Leu Ile Phe Ser ArgArg Lys Pro Ser Val Met 980 985 990 Thr Pro Ile Glu Gly Leu Asp Thr GlyGlu Met Ser Asn Ser Thr Ser 995 1000 1005 Ser Leu Lys Arg Gln Arg LeuGly Ser Glu Arg Ala Ala Ser His 1010 1015 1020 Val Ala Gln Ala Asn LeuLys Leu Leu Asp Val Ser Lys Ile Phe 1025 1030 1035 Pro Ile Ala Glu IleAla Glu Glu Ser Ser Pro Glu Val Val Pro 1040 1045 1050 Val Glu Leu LeuCys Val Pro Ser Pro Ala Ser Gln Gly Asp Leu 1055 1060 1065 His Thr LysPro Leu Gly Thr Asp Asp Asp Phe Trp Gly Pro Thr 1070 1075 1080 Gly ProVal Ala Thr Glu Val Val Asp Lys Glu Lys Asn Leu Tyr 1085 1090 1095 ArgVal His Phe Pro Val Ala Gly Ser Tyr Arg Trp Pro Asn Thr 1100 1105 1110Gly Leu Cys Phe Val Val Arg Glu Ala Val Thr Val Glu Ile Glu 1115 11201125 Phe Cys Val Trp Asp Gln Phe Leu Gly Glu Ile Asn Pro Gln His 11301135 1140 Ser Trp Met Val Ala Gly Pro Leu Leu Asp Ile Lys Ala Glu Pro1145 1150 1155 Gly Ala Val Glu Ala Val His Leu Pro His Phe Val Ala LeuGln 1160 1165 1170 Gly Gly His Val Asp Thr Ser Leu Phe Gln Val Ala HisPhe Lys 1175 1180 1185 Glu Glu Gly Met Leu Leu Glu Lys Pro Ala Arg ValGlu Leu His 1190 1195 1200 His Ile Val Leu Glu Asn Pro Ser Phe Ser ProLeu Gly Val Leu 1205 1210 1215 Leu Lys Met Ile His Asn Ala Leu Arg PheIle Pro Val Thr Ser 1220 1225 1230 Val Val Leu Leu Tyr His Arg Leu HisPro Glu Glu Val Thr Phe 1235 1240 1245 His Leu Tyr Leu Ile Pro Ser AspCys Ser Ile Arg Lys Glu Leu 1250 1255 1260 Glu Leu Cys Tyr Arg Ser ProGly Glu Asp Gln Leu Phe Ser Glu 1265 1270 1275 Phe Tyr Val Gly His LeuGly Ser Gly Ile Arg Leu Gln Val Lys 1280 1285 1290 Asp Lys Lys Asp GluThr Leu Val Trp Glu Ala Leu Val Lys Pro 1295 1300 1305 Gly Asp Leu MetPro Ala Thr Thr Leu Ile Pro Pro Ala Cys Ile 1310 1315 1320 Ala Val ProSer Pro Leu Asp Ala Pro Gln Leu Leu His Phe Val 1325 1330 1335 Asp GlnTyr Arg Glu Gln Leu Ile Ala Arg Val Thr Ser Val Glu 1340 1345 1350 ValVal Leu Asp Lys Leu His Gly Gln Val Leu Ser Gln Glu Gln 1355 1360 1365Tyr Glu Arg Val Leu Ala Glu Asn Thr Arg Pro Ser Gln Met Arg 1370 13751380 Lys Leu Phe Ser Leu Ser Gln Ser Trp Asp Arg Lys Cys Lys Asp 13851390 1395 Gly Leu Tyr Gln Ala Leu Lys Glu Thr His Pro His Leu Ile Met1400 1405 1410 Glu Leu Trp Glu Lys Gly Ser Lys Lys Gly Leu Leu Pro LeuSer 1415 1420 1425 Ser 4 1033 PRT Homo sapiens 4 Met Gly Phe Asn Leu GlnAla Leu Leu Glu Gln Leu Ser Gln Asp Glu 1 5 10 15 Leu Ser Lys Phe LysTyr Leu Ile Thr Thr Phe Ser Pro Ala His Glu 20 25 30 Leu Gln Lys Ile ProHis Lys Glu Val Asp Lys Ala Asp Gly Lys Gln 35 40 45 Leu Val Glu Ile LeuThr Thr His Cys Asp Ser Tyr Trp Val Glu Met 50 55 60 Ala Ser Leu Gln ValPhe Glu Lys Met His Arg Met Asp Leu Ser Glu 65 70 75 80 Arg Ala Lys AspGlu Val Arg Glu Ala Ala Leu Lys Ser Phe Asn Lys 85 90 95 Arg Lys Pro LeuSer Leu Gly Ile Thr Arg Lys Glu Arg Pro Pro Leu 100 105 110 Asp Val AspGlu Met Leu Glu Arg Phe Lys Thr Glu Ala Gln Asp Lys 115 120 125 Asp AsnArg Cys Arg Tyr Ile Leu Lys Thr Lys Phe Arg Glu Met Trp 130 135 140 LysSer Trp Pro Gly Asp Ser Lys Glu Val Gln Val Met Ala Glu Arg 145 150 155160 Tyr Lys Met Leu Ile Pro Phe Ser Asn Pro Arg Val Leu Pro Gly Pro 165170 175 Phe Ser Tyr Thr Val Val Leu Tyr Gly Pro Ala Gly Leu Gly Lys Thr180 185 190 Thr Leu Ala Gln Lys Leu Met Leu Asp Trp Ala Glu Asp Asn LeuIle 195 200 205 His Lys Phe Lys Tyr Ala Phe Tyr Leu Ser Cys Arg Glu LeuSer Arg 210 215 220 Leu Gly Pro Cys Ser Phe Ala Glu Leu Val Phe Arg AspTrp Pro Glu 225 230 235 240 Leu Gln Asp Asp Ile Pro His Ile Leu Ala GlnAla Arg Lys Ile Leu 245 250 255 Phe Val Ile Asp Gly Phe Asp Glu Leu GlyAla Ala Pro Gly Ala Leu 260 265 270 Ile Glu Asp Ile Cys Gly Asp Trp GluLys Lys Lys Pro Val Pro Val 275 280 285 Leu Leu Gly Ser Leu Leu Asn ArgVal Met Leu Pro Lys Ala Ala Leu 290 295 300 Leu Val Thr Thr Arg Pro ArgAla Leu Arg Asp Leu Arg Ile Leu Ala 305 310 315 320 Glu Glu Pro Ile TyrIle Arg Val Glu Gly Phe Leu Glu Glu Asp Lys 325 330 335 Arg Ala Tyr PheLeu Arg His Phe Gly Asp Glu Asp Gln Ala Met Arg 340 345 350 Ala Phe GluLeu Met Arg Ser Asn Ala Ala Leu Phe Gln Leu Gly Ser 355 360 365 Ala ProAla Val Cys Trp Ile Val Cys Thr Thr Leu Lys Leu Gln Met 370 375 380 GluLys Gly Glu Asp Pro Val Pro Thr Cys Leu Thr Arg Thr Gly Leu 385 390 395400 Phe Leu Arg Phe Leu Cys Ser Arg Phe Pro Gln Gly Ala Gln Leu Arg 405410 415 Gly Ala Leu Arg Thr Leu Ser Leu Leu Ala Ala Gln Gly Leu Trp Ala420 425 430 Gln Thr Ser Val Leu His Arg Glu Asp Leu Glu Arg Leu Gly ValGln 435 440 445 Glu Ser Asp Leu Arg Leu Phe Leu Asp Gly Asp Ile Leu ArgGln Asp 450 455 460 Arg Val Ser Lys Gly Cys Tyr Ser Phe Ile His Leu SerPhe Gln Gln 465 470 475 480 Phe Leu Thr Ala Leu Phe Tyr Thr Leu Glu LysGlu Glu Glu Glu Asp 485 490 495 Arg Asp Gly His Thr Trp Asp Ile Gly AspVal Gln Lys Leu Leu Ser 500 505 510 Gly Val Glu Arg Leu Arg Asn Pro AspLeu Ile Gln Ala Gly Tyr Tyr 515 520 525 Ser Phe Gly Leu Ala Asn Glu LysArg Ala Lys Glu Leu Glu Ala Thr 530 535 540 Phe Gly Cys Arg Met Ser ProAsp Ile Lys Gln Glu Leu Leu Arg Cys 545 550 555 560 Asp Ile Ser Cys LysGly Gly His Ser Thr Val Thr Asp Leu Gln Glu 565 570 575 Leu Leu Gly CysLeu Tyr Glu Ser Gln Glu Glu Glu Leu Val Lys Glu 580 585 590 Val Met AlaGln Phe Lys Glu Ile Ser Leu His Leu Asn Ala Val Asp 595 600 605 Val ValPro Ser Ser Phe Cys Val Lys His Cys Arg Asn Leu Gln Lys 610 615 620 MetSer Leu Gln Val Ile Lys Glu Asn Leu Pro Glu Asn Val Thr Ala 625 630 635640 Ser Glu Ser Asp Ala Glu Val Glu Arg Ser Gln Asp Asp Gln His Met 645650 655 Leu Pro Phe Trp Thr Asp Leu Cys Ser Ile Phe Gly Ser Asn Lys Asp660 665 670 Leu Met Gly Leu Ala Ile Asn Asp Ser Phe Leu Ser Ala Ser LeuVal 675 680 685 Arg Ile Leu Cys Glu Gln Ile Ala Ser Asp Thr Cys His LeuGln Arg 690 695 700 Val Val Phe Lys Asn Ile Ser Pro Ala Asp Ala His ArgAsn Leu Cys 705 710 715 720 Leu Ala Leu Arg Gly His Lys Thr Val Thr TyrLeu Thr Leu Gln Gly 725 730 735 Asn Asp Gln Asp Asp Met Phe Pro Ala LeuCys Glu Val Leu Arg His 740 745 750 Pro Glu Cys Asn Leu Arg Tyr Leu GlyLeu Val Ser Cys Ser Ala Thr 755 760 765 Thr Gln Gln Trp Ala Asp Leu SerLeu Ala Leu Glu Val Asn Gln Ser 770 775 780 Leu Thr Cys Val Asn Leu SerAsp Asn Glu Leu Leu Asp Glu Gly Ala 785 790 795 800 Lys Leu Leu Tyr ThrThr Leu Arg His Pro Lys Cys Phe Leu Gln Arg 805 810 815 Leu Ser Leu GluAsn Cys His Leu Thr Glu Ala Asn Cys Lys Asp Leu 820 825 830 Ala Ala ValLeu Val Val Ser Arg Glu Leu Thr His Leu Cys Leu Ala 835 840 845 Lys AsnPro Ile Gly Asn Thr Gly Val Lys Phe Leu Cys Glu Gly Leu 850 855 860 ArgTyr Pro Glu Cys Lys Leu Gln Thr Leu Val Leu Trp Asn Cys Asp 865 870 875880 Ile Thr Ser Asp Gly Cys Cys Asp Leu Thr Lys Leu Leu Gln Glu Lys 885890 895 Ser Ser Leu Leu Cys Leu Asp Leu Gly Leu Asn His Ile Gly Val Lys900 905 910 Gly Met Lys Phe Leu Cys Glu Ala Leu Arg Lys Pro Leu Cys AsnLeu 915 920 925 Arg Cys Leu Trp Leu Trp Gly Cys Ser Ile Pro Pro Phe SerCys Glu 930 935 940 Asp Leu Cys Ser Ala Leu Ser Asn Gln Ser Leu Val ThrLeu Asp Leu 945 950 955 960 Gly Gln Asn Pro Leu Gly Ser Ser Gly Val LysMet Leu Phe Glu Thr 965 970 975 Leu Thr Cys Ser Ser Gly Thr Leu Arg ThrLeu Arg Leu Lys Ile Asp 980 985 990 Asp Phe Asn Asp Glu Leu Asn Lys LeuLeu Glu Glu Ile Glu Glu Lys 995 1000 1005 Asn Pro Gln Leu Ile Ile AspThr Glu Lys His His Pro Trp Ala 1010 1015 1020 Glu Arg Pro Ser Ser HisAsp Phe Met Ile 1025 1030 5 2763 DNA Homo Sapiens 5 cggacgcgtgggcgcgcagc ctggctgacc tgatcctgga ccagtgcccc gaccgcggcg 60 cgccggtgccgcagatgctg gcccagccgc agcggctgct cttcatcctg gacggcgcgg 120 acgagctgccggcgctgggg ggccccgagg ccgcgccctg cacagacccc ttcgaggcgg 180 cgagcggcgcgcgggtgcta ggcgggctgc tgagtaaggc gctgctgccc acggccctcc 240 tgctggtgaccacgcgcgcc gccgcccccg ggaggctgca gggccgcctg tgttccccgc 300 agtgcgccgaggtgcgcggc ttctccgaca aggacaagaa gaagtatttc tacaagttct 360 tccgggatgagaggagggcc gagcgcgcct accgcttcgt gaaggagaac gagacgctgt 420 tcgcgctgtgcttcgtgccc ttcgtgtgct ggatcgtgtg caccgtgctg cgccagcagc 480 tggagctcggtcgggacctg tcgcgcacgt ccaagaccac cacgtcagtg tacctgcttt 540 tcatcaccagcgttctgagc tcggctccgg tagccgacgg gccccggttg cagggcgacc 600 tgcgcaatctgtgccgcctg gcccgcgagg gcgtcctcgg acgcagggcg cagtttgccg 660 agaaggaactggagcaactg gagcttcgtg gctccaaagt gcagacgctg tttctcagca 720 aaaaggagctgccgggcgtg ctggagacag aggtcaccta ccagttcatc gaccagagct 780 tccaggagttcctcgcggca ctgtcctacc tgctggagga cggcggggtg cccaggaccg 840 cggctggcggcgttgggaca ctcctgcgtg gggacgccca gccgcacagc cacttggtgc 900 tcaccacgcgcttcctcttc ggactgctga gcgcggagcg gatgcgcgac atcgagcgcc 960 acttcggctgcatggtttca gagcgtgtga agcaggaggc cctgcggtgg gtgcagggac 1020 agggacagggctgccccgga gtggcaccag aggtgaccga gggggccaaa gggctcgagg 1080 acaccgaagagccagaggag gaggaggagg gagaggagcc caactaccca ctggagttgc 1140 tgtactgcctgtacgagacg caggaggacg cgtttgtgcg ccaagccctg tgccggttcc 1200 cggagctggcgctgcagcga gtgcgcttct gccgcatgga cgtggctgtt ctgagctact 1260 gcgtgaggtgctgccctgct ggacaggcac tgcggctgat cagctgcaga ttggttgctg 1320 cgcaggagaagaagaagaag agcctgggga agcggctcca ggccagcctg ggtggcggca 1380 gttctcaaggcaccacaaaa caactgccag cctcccttct tcatccactc tttcaggcaa 1440 tgactgacccactgtgccat ctgagcagcc tcacgctgtc ccactgcaaa ctccctgacg 1500 cggtctgccgagacctttct gaggccctga gggcagcccc cgcactgacg gagctgggcc 1560 tcctccacaacaggctcagt gaggcgggac tgcgtatgct gagtgagggc ctagcctggc 1620 cgcagtgcagggtgcagacg gtcaggtgag gcctggcctg ggagggaccg tgggatgccc 1680 ccgccaccccagcagctcct gaggtcggcc ctcccacagg gtacagctgc ctgaccccca 1740 gcgagggctccagtacctgg tgggtatgct tcggcagagc cccgccctga ccaccctgga 1800 tctcagcggctgccaactgc ccgcccccat ggtgacctac ctgtgtgcag tcctgcagca 1860 ccagggatgcggcctgcaga ccctcagtct ggcctctgtg gagctgagcg agcagtcact 1920 acaggagcttcaggctgtga agagagcaaa gccggatctg gtcatcacac acccagcgct 1980 ggacggccacccacaacctc ccaaggaact catctcgacc ttctgaggct ctggtggcca 2040 gagcagggtggaagacccta gtcaaagtcc ctgtggagag aacggcccat tccaagggca 2100 ggaggatattgctctcggcc tttgggaaac ttttgagccg agaggccgca gacaggcatg 2160 tgggaggcccagacacggca ccctgccccg tccaggacag gcccaggacc tgcccctctc 2220 tccacacctggggtacccct tctcccccag ccccaccact actccaccca ccttcctctc 2280 ctgagaccctccagccattc cccttgaaaa caccccccga ccccaagcca caataatgac 2340 agcgagagctccaattaact aagcacctac ctggcggcag aataaccctt cactgcctga 2400 tccccatctgcagtgtggcc caacagcccc cagaactatg cccacataga ctggaggtag 2460 gcagttcaccgtccctccct gttaggaatg agaccatccc tgaggctatg gcccaggccc 2520 acaggcgtccagtgtctgag atctttggga agggagacta gggcaggtgg agacagcgca 2580 gaacccccgtgctgggtggg aagcatgacc acatggtggg tgagcagccc ccatgcactg 2640 acggtaaattcccctgtgga ctcatttctg ttggtttcta ttacacctgg ccaggcgtgg 2700 tacaatacaggtcggtgctc acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760 aaa 2763 62054 DNA Homo Sapiens 6 ggaactggag caactggagc ttcgtggctc caaagtgcagacgctgtttc tcagcaaaaa 60 ggagctgccg ggcgtgctgg agacagaggt cacctaccagttcatcgacc agagcttcca 120 ggagttcctc gcggcactgt cctacctgct ggaggacggcggggtgccca ggaccgcggc 180 tggcggcgtt gggacactcc tgcgtgggga cgcccagccgcacagccact tggtgctcac 240 cacgcgcttc ctcttcggac tgctgagcgc ggagcggatgcgcgacatcg agcgccactt 300 cggctgcatg gtttcagagc gtgtgaagca ggaggccctgcggtgggtgc agggacaggg 360 acagggctgc cccggagtgg caccagaggt gaccgagggggccaaagggc tcgaggacac 420 cgaagagcca gaggaggagg aggagggaga ggagcccaactacccactgg agttgctgta 480 ctgcctgtac gagacgcagg aggacgcgtt tgtgcgccaagccctgtgcc ggttcccgga 540 gctggcgctg cagcgagtgc gcttctgccg catggacgtggctgttctga gctactgcgt 600 gaggtgctgc cctgctggac aggcactgcg gctgatcagctgcagattgg ttgctgcgca 660 ggagaagaag aagaagagcc tggggaagcg gctccaggccagcctgggtg gcggcagttc 720 tcaaggcacc acaaaacaac tgccagcctc ccttcttcatccactctttc aggcaatgac 780 tgacccactg tgccatctga gcagcctcac gctgtcccactgcaaactcc ctgacgcggt 840 ctgccgagac ctttctgagg ccctgagggc agcccccgcactgacggagc tgggcctcct 900 ccacaacagg ctcagtgagg cgggactgcg tatgctgagtgagggcctag cctggccgca 960 gtgcagggtg cagacggtca gggtacagct gcctgacccccagcgagggc tccagtacct 1020 ggtgggtatg cttcggcaga gccccgccct gaccaccctggatctcagcg gctgccaact 1080 gcccgccccc atggtgacct acctgtgtgc agtcctgcagcaccagggat gcggcctgca 1140 gaccctcagt ctggcctctg tggagctgag cgagcagtcactacaggagc ttcaggctgt 1200 gaagagagca aagccggatc tggtcatcac acacccagcgctggacggcc acccacaacc 1260 tcccaaggaa ctcatctcga ccttctgagg ctctggtggccagagcaggg tggaagaccc 1320 tagtcaaagt ccctgtggag agaacggccc attccaagggcaggaggata ttgctctcgg 1380 cctttgggaa acttttgagc cgagaggccg cagacaggcatgtgggaggc ccagacacgg 1440 caccctgccc cgtccaggac aggcccagga cctgcccctctctccacacc tggggtaccc 1500 cttctccccc agccccacca ctactccacc caccttcctctcctgagacc ctccagccat 1560 tccccttgaa aacacccccc gaccccaagc cacaataatgacagcgagag ctccaattaa 1620 ctaagcacct acctgggggc agaataaccc ttcactgcctgatccccatc tgcagtgtgg 1680 cccaacagcc cccagaacta tgcccacata gactggaggtaggcagttca ccgtccctcc 1740 ctgttaggaa tgagaccatc cctgaggcta tggcccaggcccacaggcgt ccagtgtctg 1800 agatctttgg gaagggagac tagggcaggt ggagacagcgcagaaccccc gtgctgggtg 1860 ggaagcatga ccacacggtg ggtgagcagc ccccatgcactgatggtaaa ttcccctgtg 1920 gactcatttc tgttggtttc tattacacct ggccaggcgtggtacaatac aggtcggtgc 1980 tcacaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2040 aaaaaaaaaa aaaa 2054 7 314 DNA homo sapiensmisc_feature (198)..(229) wherein “n” is equal to A, C, G, or T. 7gccacttggt gctcaccacg cgcttcctct tcggactgct gagcgcggag ggatgcgcga 60catcgagcgc cacttcggct gcatggtttc agagcgtgtg aagcaggagg ccctgcggtg 120ggtgcaggga cagggacagg gctgccccgg agtggcacca gaggtgaccg agggggccaa 180agggctcgag gacaccgnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc ccaactaccc 240actggagttg ctgtactgcc tgtacgagac gcaggaggac gcgtttgtnc gccaaagccc 300tgtgccggtt cccg 314 8 24 PRT homo sapiens 8 Gly Ala Arg Val Leu Gly GlyLeu Leu Ser Lys Ala Leu Leu Pro Thr 1 5 10 15 Ala Leu Leu Leu Val ThrThr Arg 20 9 17 PRT homo sapiens 9 Leu Phe Ala Leu Cys Phe Val Pro PheVal Cys Trp Ile Val Cys Thr 1 5 10 15 Val 10 17 PRT homo sapiens 10 SerVal Tyr Leu Leu Phe Ile Thr Ser Val Leu Ser Ser Ala Pro Val 1 5 10 15Ala 11 21 DNA Homo sapiens 11 catggtttca gagcgtgtga a 21 12 23 DNA Homosapiens 12 tcgtacaggc agtacagcaa ctc 23 13 80 DNA Homo sapiens 13cttcacacgc tctgaaacca tgcagccgaa gtggcgctcg atgtcgcgca tccctccgcg 60ctcagcagtc cgaagaggaa 80 14 14 PRT homo sapiens 14 Arg Phe Val Lys GluAsn Glu Thr Leu Phe Ala Leu Cys Phe 1 5 10 15 17 PRT homo sapiens 15 PhePhe Arg Asp Glu Arg Arg Ala Glu Arg Ala Tyr Arg Phe Val Lys 1 5 10 15Glu 16 13 PRT homo sapiens 16 Ala Leu Leu Leu Val Thr Thr Arg Ala AlaAla Pro Gly 1 5 10 17 13 PRT homo sapiens 17 Glu Val Arg Gly Phe Ser AspLys Asp Lys Lys Lys Tyr 1 5 10 18 13 PRT homo sapiens 18 Arg Asp Leu SerArg Thr Ser Lys Thr Thr Thr Ser Val 1 5 10 19 13 PRT homo sapiens 19 GlnThr Leu Phe Leu Ser Lys Lys Glu Leu Pro Gly Val 1 5 10 20 13 PRT homosapiens 20 Ser His Leu Val Leu Thr Thr Arg Phe Leu Phe Gly Leu 1 5 10 2113 PRT homo sapiens 21 Phe Gly Cys Met Val Ser Glu Arg Val Lys Gln GluAla 1 5 10 22 13 PRT homo sapiens 22 Ala Leu Arg Leu Ile Ser Cys Arg LeuVal Ala Ala Gln 1 5 10 23 13 PRT homo sapiens 23 Gly Ser Ser Gln Gly ThrThr Lys Gln Leu Pro Ala Ser 1 5 10 24 13 PRT homo sapiens 24 Gln Cys ArgVal Gln Thr Val Arg Val Gln Leu Pro Asp 1 5 10 25 514 PRT homo sapiens25 Met Cys Phe Ile Pro Leu Val Cys Trp Ile Val Cys Thr Gly Leu Lys 1 510 15 Gln Gln Met Glu Ser Gly Lys Ser Leu Ala Gln Thr Ser Lys Thr Ser 2025 30 Thr Ala Val Tyr Val Phe Phe Leu Ser Ser Leu Leu Gln Pro Arg Gly 3540 45 Gly Ser Gln Glu His Gly Leu Cys Ala His Leu Trp Gly Leu Cys Ser 5055 60 Leu Ala Ala Asp Gly Ile Trp Asn Gln Lys Ile Leu Phe Glu Glu Ser 6570 75 80 Asp Leu Arg Asn His Gly Leu Gln Lys Ala Asp Val Ser Ala Phe Leu85 90 95 Arg Met Asn Leu Phe Gln Lys Glu Val Asp Cys Glu Lys Phe Tyr Ser100 105 110 Phe Ile His Met Thr Phe Gln Glu Phe Phe Ala Ala Met Tyr TyrLeu 115 120 125 Leu Glu Glu Glu Lys Glu Gly Arg Thr Asn Val Pro Gly SerArg Leu 130 135 140 Lys Leu Pro Ser Arg Asp Val Thr Val Leu Leu Glu AsnTyr Gly Lys 145 150 155 160 Phe Glu Lys Gly Tyr Leu Ile Phe Val Val ArgPhe Leu Phe Gly Leu 165 170 175 Val Asn Gln Glu Arg Thr Ser Tyr Leu GluLys Lys Leu Ser Cys Met 180 185 190 Ile Ser Gln Gln Ile Arg Leu Glu LeuLeu Lys Trp Ile Glu Val Lys 195 200 205 Ala Lys Ala Lys Lys Leu His AspGln Pro Ser Gln Leu Glu Leu Phe 210 215 220 Tyr Cys Leu Tyr Glu Met GlnGlu Glu Asp Phe Val Gln Arg Ala Met 225 230 235 240 Asp Tyr Phe Pro LysIle Glu Ile Asn Leu Ser Thr Arg Met Asp His 245 250 255 Met Val Ser SerPhe Cys Ile Glu Asn Cys His Arg Val Glu Ser Leu 260 265 270 Ser Leu GlyPhe Leu His Asn Met Pro Lys Glu Glu Glu Glu Glu Glu 275 280 285 Lys GluGly Arg His Leu Asp Met Val Gln Cys Val Leu Pro Ser Ser 290 295 300 SerHis Ala Ala Cys Ser His Gly Leu Gly Arg Cys Gly Leu Ser His 305 310 315320 Glu Cys Cys Phe Asp Ile Ser Leu Val Leu Ser Ser Asn Gln Lys Leu 325330 335 Val Glu Leu Asp Leu Ser Asp Asn Ala Leu Gly Asp Phe Gly Ile Arg340 345 350 Leu Leu Cys Val Gly Leu Lys His Leu Leu Cys Asn Leu Lys LysLeu 355 360 365 Trp Leu Val Asn Ser Ala Leu Arg Gln Ser Val Val Gln LeuCys Pro 370 375 380 Arg Tyr Ser Ala Leu Ile Arg Ile Ser Arg Thr Phe ThrAla Arg Gln 385 390 395 400 His Ser Arg Arg Gln Gly Ile Lys Leu Leu CysGlu Gly Leu Leu His 405 410 415 Pro Asp Cys Lys Leu Gln Val Leu Glu LeuAsp Asn Cys Asn Leu Thr 420 425 430 Ser His Cys Cys Trp Asp Leu Ser ThrLeu Leu Thr Ser Ser Gln Ser 435 440 445 Leu Arg Lys Leu Ser Leu Gly AsnAsn Asp Leu Gly Asp Leu Gly Val 450 455 460 Met Met Phe Cys Glu Val LeuLys Gln Gln Ser Cys Leu Leu Gln Asn 465 470 475 480 Leu Gly Leu Ser GluMet Tyr Phe Asn Tyr Glu Thr Lys Ser Ala Leu 485 490 495 Glu Thr Leu GlnGlu Glu Lys Pro Glu Leu Thr Val Val Phe Glu Pro 500 505 510 Ser Trp 261429 PRT homo sapiens 26 Met Ala Gly Gly Ala Trp Gly Arg Leu Ala Cys TyrLeu Glu Phe Leu 1 5 10 15 Lys Lys Glu Glu Leu Lys Glu Phe Gln Leu LeuLeu Ala Asn Lys Ala 20 25 30 His Ser Arg Ser Ser Ser Gly Glu Thr Pro AlaGln Pro Glu Lys Thr 35 40 45 Ser Gly Met Glu Val Ala Ser Tyr Leu Val AlaGln Tyr Gly Glu Gln 50 55 60 Arg Ala Trp Asp Leu Ala Leu His Thr Trp GluGln Met Gly Leu Arg 65 70 75 80 Ser Leu Cys Ala Gln Ala Gln Glu Gly AlaGly His Ser Pro Ser Phe 85 90 95 Pro Tyr Ser Pro Ser Glu Pro His Leu GlySer Pro Ser Gln Pro Thr 100 105 110 Ser Thr Ala Val Leu Met Pro Trp IleHis Glu Leu Pro Ala Gly Cys 115 120 125 Thr Gln Gly Ser Glu Arg Arg ValLeu Arg Gln Leu Pro Asp Thr Ser 130 135 140 Gly Arg Arg Trp Arg Glu IleSer Ala Ser Leu Leu Tyr Gln Ala Leu 145 150 155 160 Pro Ser Ser Pro AspHis Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala 165 170 175 Pro Thr Ser ThrAla Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro 180 185 190 Ser Leu AlaPro Arg Glu Gln Glu Ala Pro Gly Thr Gln Trp Pro Leu 195 200 205 Asp GluThr Ser Gly Ile Tyr Tyr Thr Glu Ile Arg Glu Arg Glu Arg 210 215 220 GluLys Ser Glu Lys Gly Arg Pro Pro Trp Ala Ala Val Val Gly Thr 225 230 235240 Pro Pro Gln Ala His Thr Ser Leu Gln Pro His His His Pro Trp Glu 245250 255 Pro Ser Val Arg Glu Ser Leu Cys Ser Thr Trp Pro Trp Lys Asn Glu260 265 270 Asp Phe Asn Gln Lys Phe Thr Gln Leu Leu Leu Leu Gln Arg ProHis 275 280 285 Pro Arg Ser Gln Asp Pro Leu Val Lys Arg Ser Trp Pro AspTyr Val 290 295 300 Glu Glu Asn Arg Gly His Leu Ile Glu Ile Arg Asp LeuPhe Gly Pro 305 310 315 320 Gly Leu Asp Thr Gln Glu Pro Arg Ile Val IleLeu Gln Gly Ala Ala 325 330 335 Gly Ile Gly Lys Ser Thr Leu Ala Arg GlnVal Lys Glu Ala Trp Gly 340 345 350 Arg Gly Gln Leu Tyr Gly Asp Arg PheGln His Val Phe Tyr Phe Ser 355 360 365 Cys Arg Glu Leu Ala Gln Ser LysVal Val Ser Leu Ala Glu Leu Ile 370 375 380 Gly Lys Asp Gly Thr Ala ThrPro Ala Pro Ile Arg Gln Ile Leu Ser 385 390 395 400 Arg Pro Glu Arg LeuLeu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405 410 415 Trp Val Leu GlnGlu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln 420 425 430 Pro Gln ProAla Asp Ala Leu Leu Gly Ser Leu Leu Gly Lys Thr Ile 435 440 445 Leu ProGlu Ala Ser Phe Leu Ile Thr Ala Arg Thr Thr Ala Leu Gln 450 455 460 AsnLeu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val Glu Val Leu Gly 465 470 475480 Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485490 495 Glu Arg Gln Ala Ile Arg Ala Phe Arg Leu Val Lys Ser Asn Lys Glu500 505 510 Leu Trp Ala Leu Cys Leu Val Pro Trp Val Ser Trp Leu Ala CysThr 515 520 525 Cys Leu Met Gln Gln Met Lys Arg Lys Glu Lys Leu Thr LeuThr Ser 530 535 540 Lys Thr Thr Thr Thr Leu Cys Leu His Tyr Leu Ala GlnAla Leu Gln 545 550 555 560 Ala Gln Pro Leu Gly Pro Gln Leu Arg Asp LeuCys Ser Leu Ala Ala 565 570 575 Glu Gly Ile Trp Gln Lys Lys Thr Leu PheSer Pro Asp Asp Leu Arg 580 585 590 Lys His Gly Leu Asp Gly Ala Ile IleSer Thr Phe Leu Lys Met Gly 595 600 605 Ile Leu Gln Glu His Pro Ile ProLeu Ser Tyr Ser Phe Ile His Leu 610 615 620 Cys Phe Gln Glu Phe Phe AlaAla Met Ser Tyr Val Leu Glu Asp Glu 625 630 635 640 Lys Gly Arg Gly LysHis Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr 645 650 655 Leu Glu Ala TyrGly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg 660 665 670 Phe Leu LeuGly Leu Leu Ser Asp Glu Gly Glu Arg Glu Met Glu Asn 675 680 685 Ile PheHis Cys Arg Leu Ser Gln Gly Arg Asn Leu Met Gln Trp Val 690 695 700 ProSer Leu Gln Leu Leu Leu Gln Pro His Ser Leu Glu Ser Leu His 705 710 715720 Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe Leu Thr Gln Val Met Ala 725730 735 His Phe Glu Glu Met Gly Met Cys Val Glu Thr Asp Met Glu Leu Leu740 745 750 Val Cys Thr Phe Cys Ile Lys Phe Ser Arg His Val Lys Lys LeuGln 755 760 765 Leu Ile Glu Gly Arg Gln His Arg Ser Thr Trp Ser Pro ThrMet Val 770 775 780 Val Leu Phe Arg Trp Val Pro Val Thr Asp Ala Tyr TrpGln Ile Leu 785 790 795 800 Phe Ser Val Leu Lys Val Thr Arg Asn Leu LysGlu Leu Asp Leu Ser 805 810 815 Gly Asn Ser Leu Ser His Ser Ala Val LysSer Leu Cys Lys Thr Leu 820 825 830 Arg Arg Pro Arg Cys Leu Leu Glu ThrLeu Arg Leu Ala Gly Cys Gly 835 840 845 Leu Thr Ala Glu Asp Cys Lys AspLeu Ala Phe Gly Leu Arg Ala Asn 850 855 860 Gln Thr Leu Thr Glu Leu AspLeu Ser Phe Asn Val Leu Thr Asp Ala 865 870 875 880 Gly Ala Lys His LeuCys Gln Arg Leu Arg Gln Pro Ser Cys Lys Leu 885 890 895 Gln Arg Leu GlnLeu Val Ser Cys Gly Leu Thr Ser Asp Cys Cys Gln 900 905 910 Asp Leu AlaSer Val Leu Ser Ala Ser Pro Ser Leu Lys Glu Leu Asp 915 920 925 Leu GlnGln Asn Asn Leu Asp Asp Val Gly Val Arg Leu Leu Cys Glu 930 935 940 GlyLeu Arg His Pro Ala Cys Lys Leu Ile Arg Leu Gly Leu Asp Gln 945 950 955960 Thr Thr Leu Ser Asp Glu Met Arg Gln Glu Leu Arg Ala Leu Glu Gln 965970 975 Glu Lys Pro Gln Leu Leu Ile Phe Ser Arg Arg Lys Pro Ser Val Met980 985 990 Thr Pro Thr Glu Gly Leu Asp Thr Gly Glu Met Ser Asn Ser ThrSer 995 1000 1005 Ser Leu Lys Arg Gln Arg Leu Gly Ser Glu Arg Ala AlaSer His 1010 1015 1020 Val Ala Gln Ala Asn Leu Lys Leu Leu Asp Val SerLys Ile Phe 1025 1030 1035 Pro Ile Ala Glu Ile Ala Glu Glu Ser Ser ProGlu Val Val Pro 1040 1045 1050 Val Glu Leu Leu Cys Val Pro Ser Pro AlaSer Gln Gly Asp Leu 1055 1060 1065 His Thr Lys Pro Leu Gly Thr Asp AspAsp Phe Trp Gly Pro Thr 1070 1075 1080 Gly Pro Val Ala Thr Glu Val ValAsp Lys Glu Lys Asn Leu Tyr 1085 1090 1095 Arg Val His Phe Pro Val AlaGly Ser Tyr Arg Trp Pro Asn Thr 1100 1105 1110 Gly Leu Cys Phe Val MetArg Glu Ala Val Thr Val Glu Ile Glu 1115 1120 1125 Phe Cys Val Trp AspGln Phe Leu Gly Glu Ile Asn Pro Gln His 1130 1135 1140 Ser Trp Met ValAla Gly Pro Leu Leu Asp Ile Lys Ala Glu Pro 1145 1150 1155 Gly Ala ValGlu Ala Val His Leu Pro His Phe Val Ala Leu Gln 1160 1165 1170 Gly GlyHis Val Asp Thr Ser Leu Phe Gln Met Ala His Phe Lys 1175 1180 1185 GluGlu Gly Met Leu Leu Glu Lys Pro Ala Arg Val Glu Leu His 1190 1195 1200His Ile Val Leu Glu Asn Pro Ser Phe Ser Pro Leu Gly Val Leu 1205 12101215 Leu Lys Met Ile His Asn Ala Leu Arg Phe Ile Pro Val Thr Ser 12201225 1230 Val Val Leu Leu Tyr His Arg Val His Pro Glu Glu Val Thr Phe1235 1240 1245 His Leu Tyr Leu Ile Pro Ser Asp Cys Ser Ile Arg Lys GluLeu 1250 1255 1260 Glu Leu Cys Tyr Arg Ser Pro Gly Glu Asp Gln Leu PheSer Glu 1265 1270 1275 Phe Tyr Val Gly His Leu Gly Ser Gly Ile Arg LeuGln Val Lys 1280 1285 1290 Asp Lys Lys Asp Glu Thr Leu Val Trp Glu AlaLeu Val Lys Pro 1295 1300 1305 Gly Asp Leu Met Pro Ala Thr Thr Leu IlePro Pro Ala Arg Ile 1310 1315 1320 Ala Val Pro Ser Pro Leu Asp Ala ProGln Leu Leu His Phe Val 1325 1330 1335 Asp Gln Tyr Arg Glu Gln Leu IleAla Arg Val Thr Ser Val Glu 1340 1345 1350 Val Val Leu Asp Lys Leu HisGly Gln Val Leu Ser Gln Glu Gln 1355 1360 1365 Tyr Glu Arg Val Leu AlaGlu Asn Thr Arg Pro Ser Gln Met Arg 1370 1375 1380 Lys Leu Phe Ser LeuSer Gln Ser Trp Asp Arg Lys Cys Lys Asp 1385 1390 1395 Gly Leu Tyr GlnAla Leu Lys Glu Thr His Pro His Leu Ile Met 1400 1405 1410 Glu Leu TrpGlu Lys Gly Ser Lys Lys Gly Leu Leu Pro Leu Ser 1415 1420 1425 Ser 27 8PRT bacteriophage T7 27 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 28 733 DNAhomo sapiens 28 gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgcccagcacctg 60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggacaccctcatga 120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaagaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagacaaagccgcggg 240 aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctgcaccaggact 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctcccaacccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtacaccctgcccc 420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtcaaaggcttct 480 atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaacaactacaaga 540 ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaagctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcatgaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtgcgacggccgc 720 gactctagag gat 733 29 39 DNA Homo sapiens 29 gcagcagcggccgcgacggg ccccggttgc agggcgacc 39 30 37 DNA Homo sapiens 30 gcagcagtcgacagaaggtc gagatgagtt ccttggg 37 31 39 DNA Homo sapiens 31 gcagcagcggccgcatgctg gcccagccgc agcggctgc 39 32 37 DNA Homo sapiens 32 gcagcagtcgacatccaggg tggtcagggc ggggctc 37 33 1032 PRT Homo sapiens 33 Met Ala SerThr Arg Cys Lys Arg Tyr Leu Glu Asp Leu Glu Asp Val 1 5 10 15 Asp LeuLys Lys Phe Lys Met His Leu Glu Asp Tyr Pro Pro Gln Lys 20 25 30 Gly CysIle Pro Leu Pro Arg Gly Gln Thr Glu Lys Ala Asp His Val 35 40 45 Asp LeuAla Thr Leu Met Ile Asp Phe Asn Gly Glu Glu Lys Ala Trp 50 55 60 Ala MetAla Val Trp Ile Phe Ala Ala Ile Asn Arg Arg Asp Leu Tyr 65 70 75 80 GluLys Ala Lys Arg Asp Glu Pro Lys Trp Gly Ser Asp Asn Ala Arg 85 90 95 ValSer Asn Pro Thr Val Ile Cys Gln Glu Asp Ser Ile Glu Glu Glu 100 105 110Trp Met Gly Leu Leu Glu Tyr Leu Ser Arg Ile Ser Ile Cys Lys Met 115 120125 Lys Lys Asp Tyr Arg Lys Lys Tyr Arg Lys Tyr Val Arg Ser Arg Phe 130135 140 Gln Cys Ile Glu Asp Arg Asn Ala Arg Leu Gly Glu Ser Val Ser Leu145 150 155 160 Asn Lys Arg Tyr Thr Arg Leu Arg Leu Ile Lys Glu His ArgSer Gln 165 170 175 Gln Glu Arg Glu Gln Glu Leu Leu Ala Ile Gly Lys ThrLys Thr Cys 180 185 190 Glu Ser Pro Val Ser Pro Ile Lys Met Glu Leu LeuPhe Asp Pro Asp 195 200 205 Asp Glu His Ser Glu Pro Val His Thr Val ValPhe Gln Gly Ala Ala 210 215 220 Gly Ile Gly Lys Thr Ile Leu Ala Arg LysMet Met Leu Asp Trp Ala 225 230 235 240 Ser Gly Thr Leu Tyr Gln Asp ArgPhe Asp Tyr Leu Phe Tyr Ile His 245 250 255 Cys Arg Glu Val Ser Leu ValThr Gln Arg Ser Leu Gly Asp Leu Ile 260 265 270 Met Ser Cys Cys Pro AspPro Asn Pro Pro Ile His Lys Ile Val Arg 275 280 285 Lys Pro Ser Arg IleLeu Phe Leu Met Asp Gly Phe Asp Glu Leu Gln 290 295 300 Gly Ala Phe AspGlu His Ile Gly Pro Leu Cys Thr Asp Trp Gln Lys 305 310 315 320 Ala GluArg Gly Asp Ile Leu Leu Ser Ser Leu Ile Arg Lys Lys Leu 325 330 335 LeuPro Glu Ala Ser Leu Leu Ile Thr Thr Arg Pro Val Ala Leu Glu 340 345 350Lys Leu Gln His Leu Leu Asp His Pro Arg His Val Glu Ile Leu Gly 355 360365 Phe Ser Glu Ala Lys Arg Lys Glu Tyr Phe Phe Lys Tyr Phe Ser Asp 370375 380 Glu Ala Gln Ala Arg Ala Ala Phe Ser Leu Ile Gln Glu Asn Glu Val385 390 395 400 Leu Phe Thr Met Cys Phe Ile Pro Leu Val Cys Trp Ile ValCys Thr 405 410 415 Gly Leu Lys Gln Gln Met Glu Ser Gly Lys Ser Leu AlaGln Thr Ser 420 425 430 Lys Thr Thr Thr Ala Val Tyr Val Phe Phe Leu SerSer Leu Leu Gln 435 440 445 Pro Arg Gly Gly Ser Gln Glu His Gly Leu CysAla His Leu Trp Gly 450 455 460 Leu Cys Ser Leu Ala Ala Asp Gly Ile TrpAsn Gln Lys Ile Leu Phe 465 470 475 480 Glu Glu Ser Asp Leu Arg Asn HisGly Leu Gln Lys Ala Asp Val Ser 485 490 495 Ala Phe Leu Arg Met Asn LeuPhe Gln Lys Glu Val Asp Cys Glu Lys 500 505 510 Phe Tyr Ser Phe Ile HisMet Thr Phe Gln Glu Phe Phe Ala Ala Met 515 520 525 Tyr Tyr Leu Leu GluGlu Glu Lys Glu Gly Arg Thr Asn Val Pro Gly 530 535 540 Ser Arg Leu LysLeu Pro Ser Arg Asp Val Thr Val Leu Leu Glu Asn 545 550 555 560 Tyr GlyLys Phe Glu Lys Gly Tyr Leu Ile Phe Val Val Arg Phe Leu 565 570 575 PheGly Leu Val Asn Gln Glu Arg Thr Ser Tyr Leu Glu Lys Lys Leu 580 585 590Ser Cys Lys Ile Ser Gln Gln Ile Arg Leu Glu Leu Leu Lys Trp Ile 595 600605 Glu Val Lys Ala Lys Ala Lys Lys Leu Gln Ile Gln Pro Ser Gln Leu 610615 620 Glu Leu Phe Tyr Cys Leu Tyr Glu Met Gln Glu Glu Asp Phe Val Gln625 630 635 640 Arg Ala Met Asp Tyr Phe Pro Lys Ile Glu Ile Asn Leu SerThr Arg 645 650 655 Met Asp His Met Val Ser Ser Phe Cys Ile Glu Asn CysHis Arg Val 660 665 670 Glu Ser Leu Ser Leu Gly Phe Leu His Asn Met ProLys Glu Glu Glu 675 680 685 Glu Glu Glu Lys Glu Gly Arg His Leu Asp MetVal Gln Cys Val Leu 690 695 700 Pro Ser Ser Ser His Ala Ala Cys Ser HisGly Leu Val Asn Ser His 705 710 715 720 Leu Thr Ser Ser Phe Cys Arg GlyLeu Phe Ser Val Leu Ser Thr Ser 725 730 735 Gln Ser Leu Thr Glu Leu AspLeu Ser Asp Asn Ser Leu Gly Asp Pro 740 745 750 Gly Met Arg Val Leu CysGlu Thr Leu Gln His Pro Gly Cys Asn Ile 755 760 765 Arg Arg Leu Trp LeuGly Arg Cys Gly Leu Ser His Glu Cys Cys Phe 770 775 780 Asp Ile Ser LeuVal Leu Ser Ser Asn Gln Lys Leu Val Glu Leu Asp 785 790 795 800 Leu SerAsp Asn Ala Leu Gly Asp Phe Gly Ile Arg Leu Leu Cys Val 805 810 815 GlyLeu Lys His Leu Leu Cys Asn Leu Lys Lys Leu Trp Leu Val Ser 820 825 830Cys Cys Leu Thr Ser Ala Cys Cys Gln Asp Leu Ala Ser Val Leu Ser 835 840845 Thr Ser His Ser Leu Thr Arg Leu Tyr Val Gly Glu Asn Ala Leu Gly 850855 860 Asp Ser Gly Val Ala Ile Leu Cys Glu Lys Ala Lys Asn Pro Gln Cys865 870 875 880 Asn Leu Gln Lys Leu Gly Leu Val Asn Ser Gly Leu Thr SerVal Cys 885 890 895 Cys Ser Ala Leu Ser Ser Val Leu Ser Thr Asn Gln AsnLeu Thr His 900 905 910 Leu Tyr Leu Arg Gly Asn Thr Leu Gly Asp Lys GlyIle Lys Leu Leu 915 920 925 Cys Glu Gly Leu Leu His Pro Asp Cys Lys LeuGln Val Leu Glu Leu 930 935 940 Asp Asn Cys Asn Leu Thr Ser His Cys CysTrp Asp Leu Ser Thr Leu 945 950 955 960 Leu Thr Ser Ser Gln Ser Leu ArgLys Leu Ser Leu Gly Asn Asn Asp 965 970 975 Leu Gly Asp Leu Gly Val MetMet Phe Cys Glu Val Leu Lys Gln Gln 980 985 990 Ser Cys Leu Leu Gln AsnLeu Gly Leu Ser Glu Met Tyr Phe Asn Tyr 995 1000 1005 Glu Thr Lys SerAla Leu Glu Thr Leu Gln Glu Glu Lys Pro Glu 1010 1015 1020 Leu Thr ValVal Phe Glu Pro Ser Trp 1025 1030 34 25 DNA artificial Synthesizedoligonucleotide. 34 cctctcatcc cggaagaacu uguag 25 35 25 DNA artificialSynthesized oligonucleotide. 35 ggcctcctgc uucacacgcu cugaa 25 36 25 DNAartificial Synthesized oligonucleotide. 36 aactcctgga agcucugguc gauga25 37 25 DNA artificial Synthesized oligonucleotide. 37 gtctgcactuuggagccacg aagct 25 38 25 DNA artificial Synthesized oligonucleotide. 38ttctccttca cgaagcggua ggcgc 25 39 24 DNA Homo sapiens 39 gaggatgaggagagctatga caca 24 40 22 DNA Homo sapiens 40 ccctttgcac tcataacgtc ag 2241 29 DNA Homo sapiens 41 aaacacacag tcatcatagg gcagctcgt 29 42 20 PRTArtificial Sequence Concensus Sequence. 42 Xaa Leu Xaa Xaa Leu Xaa LeuXaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Leu Xaa 20 43 24PRT Artificial Sequence Concensus Sequence. 43 Leu Xaa Xaa Leu Xaa XaaLeu Xaa Leu Xaa Xaa Asn Xaa Leu Xaa Xaa 1 5 10 15 Leu Pro Xaa Xaa XaaPhe Xaa Xaa 20 44 8 PRT Artificial Sequence Concensus Sequence. 44 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 45 8 PRT Artificial Sequence ConcensusSequence. 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5

1 to 23 (Cancelled)
 24. An isolated polypeptide consisting of an aminoacid sequence selected from the group consisting of: (a) an isolatedpolypeptide consisting of amino acids 1 to 625 of SEQ ID NO:2; and (b)an isolated polypeptide consisting of amino acids 2 to 625 of SEQ IDNO:2.
 25. The isolated polypeptide of claim 24, wherein saidpolynucleotide is (a).
 26. The isolated polypeptide of claim 24, whereinsaid polynucleotide is (b).
 27. An isolated polypeptide encoded by amember of the group consisting of: a.) the SILRR1a cDNA clone containedin ATCC Deposit No: PTA-2679; and b.) the SILRR1b cDNA clone containedin ATCC Deposit No: PTA-2674.
 28. An isolated polypeptide produced by amethod comprising: (a) culturing a recombinant host cell underconditions such that the polypeptide of claim 24 is expressed; and (b)recovering said polypeptide, wherein said recombinant host cellcomprises a vector comprising a recombinant nucleic acid moleculeconsisting of an isolated polynucleotide encoding a polypeptideconsisting of amino acids 1 to 625 of SEQ ID NO:2 or a polypeptideconsisting of amino acids 2 to 625 of SEQ ID NO:2, operably linked to apromoter.
 29. The isolated polypeptide produced by the method of claim28.
 30. An isolated polypeptide produced by a method comprising: (a)culturing a recombinant host cell under conditions such that thepolypeptide of claim 27 is expressed; and (b) recovering saidpolypeptide, wherein said recombinant host cell comprises a vectorcomprising a recombinant nucleic acid molecule consisting of the SILRR1acDNA clone contained in ATCC Deposit No: PTA-2679; or the SILRR1b cDNAclone contained in ATCC Deposit No: PTA-2674, operably linked to apromoter.
 31. The isolated polypeptide produced by the method of claim30.
 32. The isolated polypeptide of claim 24 wherein said amino acidsequence is fused to a heterologous polypeptide, wherein saidheterologous polypeptide is the Fc domain of immunoglobulin.